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The invention includes compositions comprising a selective small-molecule
inhibitor of RAD51 recombinase and a pharmaceutically acceptable carrier.
The invention further includes methods of treating or preventing cancer
in a subject, comprising the step of administering to the subject the
compositions contemplated within the invention.

[0002] This invention was made with government support under grant numbers
CA100839 and MH084119 awarded by the National Institutes of Health. The
government has certain rights in this invention.

Claims

1-12. (canceled)

13. A method of treating or preventing cancer in a subject in need
thereof, the method comprising administering to the subject a
pharmaceutical composition comprising a pharmaceutically effective amount
of a compound selected from the group consisting of Formula (1), Formula
(2), Formula (3), a salt thereof, and combinations thereof: ##STR00016##
wherein in (1): R.sup.1 and R.sup.2 are independently selected from the
group consisting of H, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 substituted
alkyl, phenyl, substituted phenyl, heteroaryl, substituted heteroaryl,
heterocyclyl, substituted heterocyclyl,
--(C.sub.1-C.sub.6)alkylene-phenyl,
--(C.sub.1-C.sub.6)alkylene-substituted phenyl,
--(C.sub.1-C.sub.6)alkylene-heteroaryl, and
--(C.sub.1-C.sub.6)alkylene-substituted heteroaryl; R.sup.3 is H,
C.sub.1-C.sub.6 alkyl, O(C.sub.1-C.sub.6 alkyl), F, Cl, Br or I; or a
salt thereof, the method further comprising administering to the subject
a treatment selected from the group consisting of (i) radiation therapy,
and (ii) a pharmaceutically effective amount of a chemotherapeutic agent;
whereby treating or preventing the cancer in the subject.

15. The method of claim 13, wherein administering to the subject of the
compound is performed at least 24 hours prior to administering to the
subject the radiation therapy or the chemotherapeutic agent.

16. The method of claim 15, wherein administering to the subject of the
compound is performed at least 12 hours prior to administering to the
subject the radiation therapy or the chemotherapeutic agent.

17. The method of claim 16, wherein administering to the subject of the
compound is performed at least 6 hours prior to administering to the
subject the radiation therapy or the chemotherapeutic agent.

18. The method of claim 17, wherein administering to the subject of the
compound is performed at least 3 hours prior to administering to the
subject the radiation therapy or the chemotherapeutic agent.

19. The method of claim 18, wherein administering to the subject of the
compound is performed at least 1 hour prior to administering to the
subject the radiation therapy or the chemotherapeutic agent.

20. The method of claim 13, wherein the composition comprising the
compound is co-administered to the subject with the radiation therapy or
the composition comprising the chemotherapeutic agent.

21. The method of claim 20, wherein the compound and the chemotherapeutic
agent are co-formulated in a pharmaceutical composition.

22. The method of claim 13, wherein the subject is a human.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional application of, and claims
priority to, U.S. patent application Ser. No. 14/001,806, filed Nov. 8,
2013, now issued as U.S. Pat. No. 9,216,177 on Dec. 22, 2015, which is
the U.S. national phase application filed under 35 U.S.C. .sctn.371
claiming benefit to PCT International Application No. PCT/US2012/025267,
filed Feb. 15, 2012, which claims priority under 35 U.S.C. .sctn.119(e)
to U.S. Provisional Application No. 61/447,410, filed Feb. 28, 2011, all
of which applications are incorporated herein by reference in their
entireties.

[0004] RAD51 recombinase (human sequence, SEQ ID NO:1), an ortholog of E.
coli RecA, is a key protein in homologous recombination in mammalian
cells. RAD51 promotes the repair of double-strand breaks, the most
harmful type of DNA lesion. Double-strand breaks are induced by various
chemical agents and ionizing radiation, and are also formed during the
repair of interstrand crosslinks. Once double-strand breaks are formed,
they are processed first by exonucleases to generate extensive ssDNA
tails (Cejka et al., 2010, Nature 467:112-16; Mimitou & Symington, 2009,
"DNA end resection: many nucleases make light work", DNA Repair (Amst)
8:983-95). Then RAD51 protein binds these ssDNA tails forming helical
nucleoprotein filaments that promote a search for homologous dsDNA
sequences (Kowalczykowski, 2008, Nature 453:463-66). Once homologous
dsDNA sequences are found, RAD51 promotes DNA strand exchange between the
ssDNA that resides within the filament and homologous dsDNA, i.e., an
invasion of ssDNA into homologous DNA duplex that results in the
displacement of the identical ssDNA from the duplex and formation of a
joint molecule. Joint molecules, key intermediates of DSB repair, provide
both the template and the primer for DNA repair synthesis that is
required for double-strand break repair (Paques & Haber, 1999, Microbiol.
Mol. Biol. Rev. 63:349-404).

[0006] RAD51 was found to be overexpressed in many tumors, including
familial BRCA1-deficient breast tumors (Raderschall et al., 2002, Cancer
Res 62:219-25; Xia et al., 1997, Mol. Cell. Biol. 17:7151-58; Maacke et
al., 2000, Intl. J. Cancer 88:907-13). Overexpression of RAD51 is thought
to rescue homologous recombination by compensating for the lack of
functional BRCA1 or other DNA repair proteins. Because RAD51
overexpression may contribute to chemoresistance and radioresistance of
human cancers (Ito et al., 2005, J. Gene Med. 7:1044-52), this protein
represents an important target for anti-cancer therapy. Identification
and use of RAD51 inhibitors may lead to development of novel combination
anticancer therapies. Since homologous recombination plays an important
role in the repair of double-strand breaks and interstrand crosslinks,
efficiency of traditional anticancer therapies, which widely use ionizing
radiation and other double-strand-breaking and intrastrand-crosslinking
agents, may be increased by inhibiting homologous recombination in cancer
cells by virtue of inhibiting the action of RAD51. Furthermore,
inhibitors that block specific activities of RAD51, like DNA strand
exchange or ATP hydrolysis, may be useful in the investigation of the
cellular functions of this protein. Recently, small molecules inhibitors
were employed in several studies to investigate the activity of RAD51 in
homologous recombination (Li et al., 2009, Biochemistry 48:6805-10;
Ishida et al., 2009, Nucl. Acids Res. 37:3367-76). However, so far no
specific inhibitors of RAD51 have been disclosed in the art.

[0007] There is a need in the art to identify novel small molecule
inhibitors of human RAD51 recombinase. The present invention fulfills
this need.

BRIEF SUMMARY OF THE INVENTION

[0008] The invention includes a pharmaceutical composition comprising a
pharmaceutically acceptable carrier and a compound of Formula (1):

[0011] In one embodiment, R.sup.3 is H, C.sub.1-C.sub.6 alkyl,
--O(C.sub.1-C.sub.6 alkyl), F or Cl. In another embodiment, R.sup.3 is H,
methyl, ethyl, methoxy or ethoxy. In yet another embodiment, R.sup.3 is
H.

[0012] In one embodiment, the compound is selected from the group
consisting of:

[0016] In one embodiment, the composition further comprises a
chemotherapeutic agent. In another embodiment, the agent is selected from
the group consisting of an alkylating agent, antimetabolite,
anthracycline, plant alkaloid, plant terpenoid, topoisomerase inhibitor,
antineoplastic agent, and combinations thereof.

[0017] The invention also includes a method of treating or preventing
cancer in a subject in need thereof. The method comprises administering
to the subject a pharmaceutical composition comprising a pharmaceutically
acceptable carrier and a pharmaceutically effective amount of a compound
selecting from the group consisting of Formula (1), Formula (2), Formula
(3), a salt thereof, and combinations thereof:

##STR00005##

wherein in (1): R.sup.1 and R.sup.2 are independently selected from the
group consisting of H, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 substituted
alkyl, phenyl, substituted phenyl, heteroaryl, substituted heteroaryl,
heterocyclyl, substituted heterocyclyl,
--(C.sub.1-C.sub.6)alkylene-phenyl,
--(C.sub.1-C.sub.6)alkylene-substituted phenyl,
--(C.sub.1-C.sub.6)alkylene-heteroaryl, and
--(C.sub.1-C.sub.6)alkylene-substituted heteroaryl; R.sup.3 is H,
C.sub.1-C.sub.6 alkyl, O(C.sub.1-C.sub.6 alkyl), F, Cl, Br or I; or a
salt thereof. The method further comprises administering to the subject a
treatment selected from the group consisting of (i) radiation therapy,
and (ii) a pharmaceutical composition comprising a pharmaceutically
effective amount of a chemotherapeutic agent; whereby treating or
preventing the cancer in the subject.

[0018] In one embodiment, the compound is selected from the group
consisting of (E)-3-benzyl-2-(2-(pyridin-3-yl)vinyl)quinazolin-4(3H)-one
(1a), (E)-3-ethyl-2-(2-(pyridin-3-yl)vinyl)quinazolin-4(3H)-one (1b),
(E)-2-(2-(pyridin-3-yl)vinyl)-3-(m-tolyl)quinazolin-4(3H)-one (1c),
1,4,10-trihydroxy-5-(hydroxymethyl)-8-methyl-3,7-dioxo-3,7-dihydro-1H-ben-
zo[6,7][1,4]dioxepino[2,3-e]isobenzofuran-11-carbaldehyde (2),
1,4-dihydroxy-10-methoxy-5,8-dimethyl-3,7-dioxo-3,7-dihydro-1H-benzo[6,7]-
[1,4]dioxepino[2,3-e]isobenzofuran-11-carbaldehyde (3), a salt thereof,
and mixtures thereof.

[0019] In one embodiment, the administering to the subject of the compound
is performed at least 24 hours prior to the administering to the subject
the radiation therapy or the chemotherapeutic agent. In another
embodiment, the administering to the subject of the compound is performed
at least 12 hours prior to the administering to the subject the radiation
therapy or the chemotherapeutic agent. In yet another embodiment, the
administering to the subject of the compound is performed at least 6
hours prior to the administering to the subject the radiation therapy or
the chemotherapeutic agent. In yet another embodiment, the administering
to the subject of the compound is performed at least 3 hours prior to the
administering to the subject the radiation therapy or the
chemotherapeutic agent. In yet another embodiment, the administering to
the subject of the compound is performed at least 1 hour prior to
administering to the subject the radiation therapy or the
chemotherapeutic agent.

[0020] In one embodiment, the composition comprising the compound is
co-administered to the subject with the radiation therapy or the
composition comprising the chemotherapeutic agent. In another embodiment,
the compound and the chemotherapeutic agent are co-formulated in a
pharmaceutical composition. In yet another embodiment, the subject is a
human.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] For the purpose of illustrating the invention, there are depicted
in the drawings certain embodiments of the invention. However, the
invention is not limited to the precise arrangements and
instrumentalities of the embodiments depicted in the drawings.

[0024] FIGS. 3A-3E illustrate the secondary screening of the RAD51
inhibitors using the D-loop assay. FIG. 3A is a scheme illustrating the
D-loop formation promoted by RAD51. The asterisk denotes the .sup.32P
label. FIG. 3B is a reproduction of an electrophoresis gel that
illustrates the analysis of 17 compounds selected by HTS. RAD51 (1 .mu.M)
was incubated with a 90-mer ssDNA (3 .mu.M) (SEQ ID NO:6) to form the
filament followed by addition of indicated compounds (100 .mu.M). Joint
molecule (D-loop) formation was initiated by addition of pUC19
supercoiled dsDNA (50 .mu.M). The DNA products were analyzed by
electrophoresis in a 1% agarose gel. The control was carried out under
identical conditions except that no tested compounds were added. FIG. 3C
is a graph illustrating the effect of selected compounds on the yield of
joint molecules. The extent of D-loop formation in the absence of
inhibitors, 40.3%, was expressed as 100% of D-loop formation efficiency.
Experiments were repeated at least three times; error bars represent
standard deviation (standard deviation). FIG. 3D comprises reproductions
of electrophoresis gels that illustrate the effect of Compound B02
concentration on the DNA strand exchange activity of RAD51 and RecA.
After incubation of RAD51 (0.3 .mu.M) or RecA (0.3 .mu.M) with Compound
B02 in indicated concentrations for 30 min, 0.9 .mu.M ssDNA (SEQ ID NO:6,
90 mer) was added to form RAD51 nucleoprotein filament for 15 min. The
D-loop formation was initiated by addition supercoiled pUC19 dsDNA (15
.mu.M). The control reactions containing no proteins are shown in lane 1
and 10. In FIG. 3E, the yield of joint molecules (D-loops) was plotted as
a graph. The extents of D-loop formation in the absence of Compound B02,
32% and 33.7% for RAD51 and RecA, respectively, were expressed as 100% of
D-loop formation efficiency. Experiments were repeated at least three
times; error bars represent S.E.M (standard error of the mean).

[0025] FIG. 4 is a graph illustrating the IC.sub.50 of RAD51 inhibition by
four selected compounds determined in the D-loop assay. RAD51 (1 .mu.M)
was incubated with a 90-mer ssDNA (3 .mu.M) (SEQ ID NO:6) to form the
filament followed by addition of Compounds A03, A04, A10, and B02 in
indicated concentrations. After a 30-min incubation, D-loop formation was
initiated by addition of pUC19 supercoiled dsDNA (50 .mu.M). The DNA
products were analyzed by electrophoresis in a 1% agarose gel.
Experiments were repeated at least three times; error bars represent
standard deviation

[0026] FIGS. 5A-5D are a series of graphs illustrating the specificity of
RAD51 inhibition by Compounds A03, A04, A10, and B02. RAD51 (1 .mu.M) or
RecA (1 .mu.M) was incubated with a 90-mer ssDNA (3 .mu.M) (SEQ ID NO:6)
for 15 min (for RAD51) or 5 min (for RecA) to form the nucleoprotein
filament. Then, tested compounds in indicated concentrations were added
and incubation continued for 30 min. The D-loop formation was initiated
by addition of pUC19 supercoiled dsDNA (50 .mu.M) and continued for 15
min (for RAD51) or 3 min (for RecA). The DNA products were analyzed by
electrophoresis in a 1% agarose gel. The yield of joint molecules
(D-loops) was plotted as a graph. Experiments were repeated at least
three times; error bars represent standard deviation.

[0027] FIGS. 6A-6C illustrate the effect of Compounds A03, A04, A10 and
B02 on branch migration activity of RAD54. FIG. 6A is a scheme that
illustrates a scheme of branch migration promoted by RAD54. The asterisk
denotes the .sup.32P label. FIGS. 6B and 6C: branch migration was
initiated by adding RAD54 (100 nM) to the mixtures containing
PX-junctions (33 nM, molecules) and the small molecule inhibitors (in
indicated concentrations). DNA products were analyzed by electrophoresis
in 8% polyacrylamide gels. For each inhibitor concentration, the extent
of branch migration was determined after 5 min of reaction (linear
phase). The results are presented as graphs in FIGS. 6B and 6C.
Experiments were repeated at least three times; error bars represent
standard deviation.

[0028] FIGS. 7A-7C illustrate the analysis of Structure-Activity
Relationship (SAR) of Compound B02. FIG. 7A illustrates the structures of
Compound B02 and its derivatives. FIG. 7B is a reproduction of an
electrophoresis gel that illustrates the effect of indicated B02
derivatives on D-loop formation by RAD51. RAD51 (1 .mu.M) was incubated
with a 90-mer ssDNA (3 .mu.M) (SEQ ID NO:6) for 15 min followed by
addition of indicated compounds (50 .mu.M). After a 30-min incubation,
the D-loop formation was initiated by addition of 50 .mu.M supercoiled
pUC19 dsDNA. The DNA products were analyzed by electrophoresis in a 1%
agarose gel. The control reaction was performed under the identical
conditions, except that no tested compounds were added. FIG. 7C is a
graph that illustrates the yield of joint molecules (D-loops).
Experiments were repeated at least three times; error bars represent
standard deviation.

[0029] FIGS. 8A-8C illustrate the effect of Compounds C3a and C3b on DNA
strand exchange activity of RAD51 and RecA. FIG. 8A is a reproduction of
an electrophoresis gel illustrating the effect of C3a on the DNA strand
exchange activity of RAD51 and RecA. The nucleoprotein filaments were
formed by incubating RAD51 (1 .mu.M) or RecA (1 .mu.M) with ssDNA (3
.mu.M), then C3a was added in indicated concentrations and incubation
continued for 30 min. D-loop formation was initiated by adding pUC19
supercoiled dsDNA (50 .mu.M). The DNA products were analyzed by
electrophoresis in a 1% agarose gel. The data from FIG. 8A was plotted as
a graph in FIG. 8B. The yield of D-loop formation in the absence of C3a
was expressed as 100%; the actual yield was 45.1% and 34.2%, for RecA and
RAD51, respectively. FIG. 8C illustrates the effect of C3b on the DNA
strand exchange activity of RAD51 and RecA. The reactions were carried
out as in FIG. 8A; the data are plotted as a graph. The yield of D-loop
formation in the absence of C3b was expressed as 100%; the actual yield
was 48.1% and 31.6%, for RecA and RAD51, respectively. Experiments were
repeated at least three times; error bars represent standard deviation

[0030] FIG. 9 is an illustration of the structure of
(E)-3-benzyl-2-(2-(pyridin-3-yl) vinyl) quinazolin-4(3H)-one) (EBVQ or
Compound B02).

[0031] FIGS. 10A-10C illustrate the inhibition by Compound B02 of the
three strand exchange promoted by RAD51 protein. FIG. 10A is a schematic
representation of DNA strand exchange between .phi.X174 circular ssDNA
and linear .phi.X174 dsDNA (linearized by ApaL1 restriction
endonuclease). FIG. 10B is a reproduction of an electrophoresis gel
illustrating the effect of the Compound B02 concentration on the
efficiency of three strand exchange assay promoted by RAD51. RAD51 was
incubated with indicated concentration of Compound B02 (lane 2-9) for 30
min; then .phi.X174 circular ssDNA and RPA were added in turn, each
addition was followed by a 5-min incubation; the strand exchange was
initiated by addition of linear .phi.X174 dsDNA. The DNA products were
analyzed by electrophoresis in a 1% agarose gel. FIG. 10C is a graph
illustrating data from FIG. 10B. Experiments were repeated at least three
times; error bars represent standard error of the mean.

[0032] FIGS. 11A-11C illustrate the finding that the order of Compound B02
addition affects the efficiency of D-loop formation promoted by RAD51.
FIG. 11A is a reproduction of an electrophoresis gel illustrating the
effect of the order of addition of Compound B02. The numbers above the
arrows indicate time of incubation. (I) Compound B02 (20 .mu.M) was added
after the RAD51-ssDNA filament formation; (II) Compound B02 (20 .mu.M)
was added to RAD51 before addition of ssDNA. FIG. 11B is a graph
illustrating the analysis of joint molecules by electrophoresis in a 1%
agarose gel. FIG. 11C is a graph in which the relative inhibition of
joint molecule formation by Compound B02 is expressed as the ratio of the
joint molecules formed by RAD51 after Compound B02 treatment to those
formed by RAD51 without Compound B02 treatment. "t" denotes the time
period between the addition of Compound B02 and dsDNA. Experiments were
repeated at least three times; error bars represent standard error of the
mean.

[0033] FIGS. 12A-12B illustrate the finding that Compound B02 inhibits
ssDNA binding of RAD51. FIG. 12A is a reproduction of an electrophoresis
gel in which RAD51 (1 .mu.M) was incubated with .sup.32P-labeled ssDNA
(SEQ ID NO:6, 90 mer) (2.5 .mu.M, nt) in buffer containing indicated NaCl
concentration either in the absence (lanes 2-7) or presence (lanes 8-13)
of Compound B02 (25 .mu.M). RAD51-ssDNA complexes were analyzed by
electrophoresis in a 10% polyacrylamide gel. The results in FIG. 12A were
shown as a graph in FIG. 12B. Lane 1 shows migration of free ssDNA.
Experiments were repeated at least three times; error bars represent
standard error of the mean.

[0035] FIGS. 14A-14B illustrate the inhibition by Compound B02 of the
coaggregation of dsDNA with the RAD51-ssDNA filament. FIG. 14A is a
scheme illustrating dsDNA coaggregation. FIG. 14B is a graph illustrating
inhibition of the coaggregation of dsDNA and RAD51-ssDNA filament by
Compound B02. To form the RAD51-ssDNA filaments, RAD51 (1 .mu.M) and
ssDNA (SEQ ID NO:7, 94mer) (3 .mu.M, nt) were incubated for 15 min. After
filament formation, NaCl was added in indicated concentrations and
coaggregation was initiated immediately by addition of .sup.32P-labeled
linear pUC19 dsDNA (linearized by BamHI restriction endonuclease) (25
.mu.M, nt). Experiments were repeated at least three times; error bars
represent standard error of the mean.

[0036] FIGS. 15A-15C illustrate the finding that Compound B02 inhibits
DSB-induced homologous recombination in human cells. FIG. 15A is a scheme
illustrating the process of measuring the frequency of homologous
recombination in human cells using the DRGFP reporter system. FIG. 15B is
a set of panels (panels 1-6) illustrating the effect of Compound B02 on
the repair of I-SceI-induced DSBs in 293 HEK cells carrying the
chromosomally located DR-GFP reporter, as determined using flow
cytometry. Green fluorescence (GRN-Hlog, indicated as "G" in the figure)
was plotted against red fluorescence (RED-Hlog, indicated as "R" in the
figure) for the sample of 10,000 cells. The GFP-positive population is
denoted by the elliptical M1 marker. Cells with I-SceI-induced DSBs were
either untreated (panel 2) or treated with Compound B02 5 .mu.M (panel
3), 10 .mu.M (panel 4), or 20 .mu.M (panel 5). As a negative control,
parental uninduced and untreated cells are shown in panel 1. As a
positive control, parental cells that were transfected with pMX-GFP
plasmid encoding GFP protein are shown in panel 6. FIG. 15C is a graph
illustrating the correlation of GFP positive cells as a function of
Compound B02 concentration. To determine the effect of Compound B02 on
the efficiency of formation of GFP-positive cells (transfection plus GFP
expression) 293 HEK cells were treated with Compound B02 in 5 .mu.M, 10
.mu.M, and 20 .mu.M concentration and then transfected with
pMX-GFP-plasmid. Data are shown as a graph (denoted as "pMX-GFP",
efficiency of formation of GFP-positive cells in the cells without
Compound B02 treatment was expressed as 100% formation efficiency) along
with the data from FIG. 15B (panels 2-5) that demonstrate the effect of
Compound B02 on the formation GFP positive cells resulted from
DSB-induced homologous recombination (denoted "I-SceI", formation of
GFP-positive cells in no Compound B02 treatment cells was expressed as
100%). Experiments were repeated at least three times; error bars
represent standard deviation.

[0037] FIGS. 16A-16D illustrate that finding that treatment with Compound
B02 does not affect the expression levels of I-SceI and RAD51 in human
cells. As illustrated in FIG. 16A, 293 HEK cells carrying the DR-GFP
reporter were treated with Compound B02 in indicated concentrations or
left untreated for 1hr; and then pCbASce plasmid expressing I-SceI was
transfected into the cells by GenDrill.TM. transfection reagent. After
incubation, during which Compound B02 was present, until cell confluence
(.about.64 h) cells were lysed and the expression level of I-SceI
restriction endonuclease containing the HA antigen was determined by
western blotting using HA-Tag antibodies. Actin that was probed with
specific antibodies was used as a quantity standard. The data from FIG.
16A is illustrated as a graph in FIG. 16B. As illustrated in FIG. 16C,
log-phase 293 HEK cells carrying the DR-GFP were incubated either in the
absence or presence of Compound B02 (20 .mu.M) until cells reached
confluence (.about.24h), the level of RAD51 expression was analyzed by
western blotting using specific antibodies against RAD51. Purified RAD51
protein (56 ng) was used as a standard. The data from FIG. 16C is
illustrated as a graph in FIG. 16D. Experiments were repeated at least
three times; error bars represent standard deviation.

[0039] FIG. 18 is a graph illustrating the finding that cotreatment of MEF
cells with a PARP-1 inhibitor AZD2281 and Compound B02 further increases
cell sensitivity to DNA damaging agents. MEF cells were treated with MMS
(.smallcircle.), and then some fractions of these cells were additionally
treated with Compound B02 (5 .mu.M) (.DELTA.), AZD2281 (1 .lamda.M)
(.smallcircle.), or with both Compound B02 (5 .mu.M) and AZD2281 (1
.mu.M) (.quadrature.). Experiments were repeated at least three times;
error bars represent standard deviation.

[0040] FIGS. 19A-19F illustrate the finding that Compound B02 interacts
with RAD51 and inhibits its activities. FIG. 19A depicts the structure of
Compound B02. FIG. 19B depicts the scheme of the DNA strand exchange and
branch migration assays. The asterisk denotes the .sup.32P label. FIG.
19C depicts the effect of Compound B02 (10 to 100 .mu.M) on the DNA
strand exchange activity of RAD51. FIG. 19D is a graph illustrating the
yield of RAD51- and RecA-generated joint molecules (JM). FIG. 19E depicts
the effect of Compound B02 (10 to 100 .mu.M) on the branch migration
activity of RAD51. Lanes 1 and 11 represent JMs before and after 8
h-incubation in the absence RAD51, respectively. FIG. 19F is a graph
illustrating the yield of the RAD51- and RecA-generated nicked circle
(NC) product. The extent of JM and NC formation in the absence of
Compound B02 was expressed as 100%; the actual extent was 32% and 15% of
JM (relative to linear dsDNA) and 21% and 63% of nicked circles for RAD51
and RecA (relative to JM-substrate), respectively. Controls containing no
RAD51 are shown in lane 1. Experiments were repeated at least three
times; error bars represent standard deviation (S.D.).

[0041] FIGS. 20A-20C illustrate the finding that Compound B02 does not
inhibit the DNA strand exchange and branch migration promoted by RecA.
FIG. 20A illustrates the scheme of the DNA strand exchange and branch
migration assays. The asterisk denotes the .sup.32P label. FIG. 20B
illustrates the effect of Compound B02 on the DNA strand exchange
activity of RecA. FIG. 20C illustrates the effect of Compound B02 on the
DNA branch migration activity of RecA. Initial DNA substrates are shown
in lane 1. Experiments were repeated at least three times; representative
gels are shown.

[0042] FIG. 21 illustrates the finding that Compound B02 binds directly to
RAD51. Compound B02 (50 .mu.M) was injected onto a sensor chip to which
RAD51 or RecA had been immobilized. The running buffer S was supplemented
with ATP (100 .mu.M). Responses to Compound B02 were normalized to the
theoretical maximum response of the surface (Rmax), assuming a 1:1
interaction. Experiments were repeated at least three times; error bars
represent S.D.

[0043] FIGS. 22A-22D illustrate the measurement of Compound B02 binding to
RAD51 using SPR. The SPR analysis was performed on a GLH high-capacity
sensor chip (Bio-Rad, Hercules, Calif.) with a high density of
immobilized RAD51 (14,000 RU) (FIGS. 22A and 22B) or RecA (9,000 RU)
(FIGS. 22C and 22D). Compound B02 at concentrations of 6.25, 12.5, 25,
and 50 .mu.M in buffer S without ATP (FIGS. 22A and 22C) or with ATP (100
.mu.M) (FIGS. 22B and 22D) was injected to the chip. A chip with the
immobilized HIV-1NL4-3 capsid protein served as a reference. Colored
lines indicate experimental data, whereas black lines indicate fitting to
the simple 1:1 binding model using the ProteOn Manager Software version
3.0 (Bio-Rad). When Compound B02 was injected over RAD51 in buffer S
containing ATP (100 .mu.M), the data did not fit to the simple binding
model, probably due to a heterogeneity in the nucleotide binding states
of the immobilized RAD51. For Compound B02 binding to RAD51 in the
absence of ATP, kinetic values are as follows:
ka=4.5(.+-.0.3).times.10.sup.3 M.sup.-1s.sup.-1 ;
kd=2.5(.+-.0.3).times.10.sup.-2s.sup.-1; Kd=5.6 .mu.M. Experiments were
repeated at least three times; numbers in parentheses represent S.D.

[0044] FIGS. 23A-23C illustrate the finding that B02 disrupts the RAD51
foci formation. FIG. 23A illustrates HEK cells which were exposed to 0.5
Gy of IR in either the presence (50 .mu.M) or the absence of B02. RAD51
foci were visualized by immunostaining using RAD51 antibodies. Nuclei
were counterstained with DAPI. Bars indicate 20 .mu.m. FIG. 23B
illustrates the fraction of foci-positive cells (the cells with .gtoreq.1
focus), which was determined by counting at least 150 cells in each
experiment. FIG. 23C illustrates the determination of the mean of foci
number per nucleus in focipositive cells by counting at least 50 cells in
each experiment. Experiments were repeated three times; error bars
represent S.D.

[0045] FIGS. 24A-24F illustrate the finding that B02 increases cell
sensitivity to DNA-damaging agents. FIGS. 24A and 24B illustrate the
survival of MEF treated with cisplatin or MMC for 1 h in the absence or
presence of B02 (5 .mu.M). FIG. 24C illustrates the effect of B02 on
survival of MEF and Tp53-/- MEF. FIG. 24D illustrates the effect of B02
(0 to 10 .mu.M) and RAD51 siRNA on HEK cells' sensitivity to cisplatin.
HEK cells were transfected with RAD51 siRNA and incubated 40 h before
treatment with cisplatin at indicated concentrations and B02 (5 .mu.M).
FIG. 24E illustrates the effect of B02 incubation time with HEK on cell
sensitivity to cisplatin. HEK cells were treated with B02 (5 .mu.M) for 1
h followed by addition of cisplatin (16 .mu.M) and incubation for 1 h.
Then cisplatin was removed and cells were incubated with B02 (5 .mu.M)
for the indicated times followed by media replacement and additional
incubation for 7-10 days. FIG. 24F illustrates the effect of AZD2281
(0.01 .mu.M) and B02 (5 .mu.M) on MEF sensitivity to MMS. Experiments
were repeated at least three times; error bars represent S.D.

[0046] FIGS. 25A-25B illustrate the finding that Compound B02 increases
the sensitivity of Tp53.sup.-/- MEF to cisplatin and MMC. Tp53.sup.-/-
MEF were treated with cisplatin (FIG. 25A) or MMC (FIG. 25B) for 1 h in
the absence or presence of B02 (5 .mu.M). Experiments were repeated at
least three times; error bars represent S.D.

[0047] FIGS. 26A-26B illustrate the effect of RAD51 siRNA on the
expression level of RAD51. FIG. 26A illustrates the RAD51 expression
levels 24, 48, 72, and 96 h after siRNA transfection determined by
Western blotting. The RAD51 protein level 48 h after transfection of HEK
cells with scrambled siRNA (denoted as "sc siRNA") was expressed as 100%.
Purified RAD51 (3.2 ng) was used as a marker. FIG. 26B is a graphical
depiction of the results of FIG. 26A.

[0048] FIG. 27 illustrates the finding that Compound B02 inhibits HR by
disrupting RAD51 binding to DNA. During the repair of DNA double-strand
breaks, RAD51 binds to ssDNA forming the nucleoprotein filament. The
filament searches for homologous dsDNA sequence to form joint molecules.
The homologous DNA then is used as a template for DNA polymerase.
Dissociation of the joint molecules and re-annealing of DNA ends lead to
the restoration of the DNA structure. Compound B02 inhibits HR by
disrupting formation of the RAD51-ssDNA filament and interaction of the
filament with dsDNA during the search for homology.

[0050] This invention includes the unexpected identification of novel
selective small-molecule RAD51 inhibitors and their utility in the
treatment of cancer. In order to identify selective RAD51 inhibitors, an
efficient high throughput screening (HTS) of chemical compound libraries
was performed using an assay based on fluorescence resonance energy
transfer (FRET). Compounds found to inhibit RAD51 DNA strand exchange
activity were further analyzed by the D-loop assay (a secondary
non-fluorescent DNA strand exchange assay), and potent RAD51 inhibitors
were thus identified.

[0051] Due to their unique mechanism, the compounds contemplated within
the invention are useful in overcoming the chemoresistance and
radioresistance of human cancers. In one aspect, treatment of a subject
with compounds contemplated within the invention enhances cell
sensitivity to radiation treatments or chemotherapeutic agents, such as
DNA cross-linking agents, cisplatin and mitomycin C. In another aspect, a
subject treated with compounds contemplated within the invention along
with a chemotherapeutic agent and/or radiation enjoys greater overall
efficacy in the cancer treatment and/or prevention, as compared to the
efficacy observed with the same dose of chemotherapeutic agent and/or
radiation alone. In yet another aspect, a subject treated with compounds
contemplated within the invention may be treated with lower doses of the
chemotherapeutic agent and/or radiation of choice, and still experience
similar efficacy in cancer treatment and/or prevention, as compared to
the standard dose of chemotherapeutic agent and/or radiation. This has
the advantage of reducing complications due to toxicity from radiation
therapy or chemotherapy, and reducing recovery times for the subject.

Definitions

[0052] As used herein, each of the following terms has the meaning
associated with it in this section.

[0053] Unless defined otherwise, all technical and scientific terms used
herein generally have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Generally, the
nomenclature used herein and the laboratory procedures in biochemistry,
analytical chemistry and organic chemistry are those well-known and
commonly employed in the art. Standard techniques or modifications
thereof are used for chemical syntheses and chemical analyses.

[0054] The articles "a" and "an" are used herein to refer to one or to
more than one (i.e. to at least one) of the grammatical object of the
article. By way of example, "an element" means one element or more than
one element.

[0055] The term "about" will be understood by persons of ordinary skill in
the art and will vary to some extent on the context in which it is used.
As used herein, "about" when referring to a measurable value such as an
amount, a temporal duration, and the like, is meant to encompass
variations of .+-.20% or .+-.10%, more preferably .+-.5%, even more
preferably .+-.1%, and still more preferably .+-.0.1% from the specified
value, as such variations are appropriate to perform the disclosed
methods.

[0056] As used herein, the term "chemotherapeutic agent" or
"chemotherapeutic agent" refers to a chemical compound, chemical
conjugate, peptide, protein, antibody and the like that finds use in
treating, preventing, or reducing the symptoms of cancer.

[0057] As used herein, the term "BHQ1" or "black hole quencher 1" refers
to the following moiety or a derivative (here, BHQ1 is illustrated as
bound to a 5'-oligo through a phosphate bond):

##STR00006##

[0058] As used herein, the terms "EBVQ", Compound B02 or Compound la are
interchangeable and refer to
(E)-3-benzyl-2-(2-(pyridin-3-yl)vinyl)quinazolin-4(3H)-one or a salt
thereof.

[0059] An "amino acid" as used herein is meant to include both natural and
synthetic amino acids, and both D and L amino acids. "Standard amino
acid" means any of the twenty L-amino acids commonly found in naturally
occurring peptides. "Nonstandard amino acid residues" means any amino
acid, other than the standard amino acids, regardless of whether it is
prepared synthetically or derived from a natural source. As used herein,
"synthetic amino acid" also encompasses chemically modified amino acids,
including but not limited to salts, amino acid derivatives (such as
amides), and substitutions. Amino acids contained within the peptides,
and particularly at the carboxy- or amino-terminus, can be modified by
methylation, amidation, acetylation or substitution with other chemical
groups which can change a peptide's circulating half-life without
adversely affecting activity of the peptide. Additionally, a disulfide
linkage may be present or absent in the peptides.

[0060] As used herein, the terms "protein", "peptide" and "polypeptide"
are used interchangeably, and refer to a compound comprised of amino acid
residues covalently linked by peptide bonds. The term "peptide bond"
means a covalent amide linkage formed by loss of a molecule of water
between the carboxyl group of one amino acid and the amino group of a
second amino acid. A protein or peptide must contain at least two amino
acids, and no limitation is placed on the maximum number of amino acids
that may comprise the sequence of a protein or peptide. Polypeptides
include any peptide or protein comprising two or more amino acids joined
to each other by peptide bonds. As used herein, the term refers to both
short chains, which also commonly are referred to in the art as peptides,
oligopeptides and oligomers, for example, and to longer chains, which
generally are referred to in the art as proteins, of which there are many
types. "Proteins" include, for example, biologically active fragments,
substantially homologous proteins, oligopeptides, homodimers,
heterodimers, protein variants, modified proteins, derivatives, analogs,
and fusion proteins, among others. The proteins include natural proteins,
recombinant proteins, synthetic proteins, or a combination thereof. A
protein may be a receptor or a non-receptor.

[0061] As used herein, amino acids are represented by the full name
thereof, by the three-letter code, as well as the one-letter code
corresponding thereto, as indicated in the following table. The structure
of amino acids and their abbreviations can also be found in the chemical
literature, such as in Stryer, 1988, "Biochemistry", 3.sup.rd Ed., W. H.
Freeman and Co., New York.

[0062] As used herein, the term "fragment," as applied to a protein or
peptide, refers to a subsequence of a larger protein or peptide. A
"fragment" of a protein or peptide may be at least about 20 amino acids
in length; for example at least about 50 amino acids in length; at least
about 100 amino acids in length, at least about 200 amino acids in
length, at least about 300 amino acids in length, and at least about 400
amino acids in length (and any integer value in between).

[0063] By "nucleic acid" is meant any nucleic acid, whether composed of
deoxyribonucleosides or ribonucleosides, and whether composed of
phosphodiester linkages or modified linkages such as phosphotriester,
phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate,
carbamate, thioether, bridged phosphoramidate, bridged methylene
phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate,
bridged phosphorothioate or sulfone linkages, and combinations of such
linkages. The term nucleic acid also specifically includes nucleic acids
composed of bases other than the five biologically occurring bases
(adenine, guanine, thymine, cytosine and uracil). The term "nucleic acid"
typically refers to large polynucleotides.

[0064] The term "DNA" as used herein is defined as deoxyribonucleic acid.

[0065] The term "RNA" as used herein is defined as ribonucleic acid.

[0066] The term "recombinant DNA" as used herein is defined as DNA
produced by joining pieces of DNA from different sources.

[0067] As used herein, the term "fragment," as applied to a nucleic acid,
refers to a subsequence of a larger nucleic acid. A "fragment" of a
nucleic acid can be at least about 15 nucleotides in length; for example,
at least about 50 nucleotides to about 100 nucleotides; at least about
100 to about 500 nucleotides, at least about 500 to about 1000
nucleotides, at least about 1000 nucleotides to about 1500 nucleotides;
or about 1500 nucleotides to about 2500 nucleotides; or about 2500
nucleotides (and any integer value in between).

[0068] Conventional notation is used herein to describe polynucleotide
sequences: the left-hand end of a single-stranded polynucleotide sequence
is the 5'-end; the left-hand direction of a double-stranded
polynucleotide sequence is referred to as the 5'-direction.

[0069] The direction of 5' to 3' addition of nucleotides to nascent RNA
transcripts is referred to as the transcription direction. The DNA strand
having the same sequence as an mRNA is referred to as the "coding
strand"; sequences on the DNA strand which are located 5' to a reference
point on the DNA are referred to as "upstream sequences"; sequences on
the DNA strand which are 3' to a reference point on the DNA are referred
to as "downstream sequences."

[0070] An "isolated nucleic acid" refers to a nucleic acid segment or
fragment which has been separated from sequences which flank it in a
naturally occurring state, i.e., a DNA fragment which has been removed
from the sequences which are normally adjacent to the fragment, i.e., the
sequences adjacent to the fragment in a genome in which it naturally
occurs. The term also applies to nucleic acids which have been
substantially purified from other components which naturally accompany
the nucleic acid, i.e., RNA or DNA or proteins, which naturally accompany
it in the cell. The term therefore includes, for example, a recombinant
DNA which is incorporated into a vector, into an autonomously replicating
plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote,
or which exists as a separate molecule (i.e., as a cDNA or a genomic or
cDNA fragment produced by PCR or restriction enzyme digestion)
independent of other sequences. It also includes a recombinant DNA which
is part of a hybrid gene encoding additional polypeptide sequence.

[0071] In the context of the present invention, the following
abbreviations for the commonly occurring nucleic acid bases are used. "A"
refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T"
refers to thymidine, and "U" refers to uridine.

[0072] The term "oligonucleotide" typically refers to short
polynucleotides, generally no greater than about 60 nucleotides. It will
be understood that when a nucleotide sequence is represented by a DNA
sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A,
U, G, C) in which "U" replaces "T."

[0073] The term "polynucleotide" as used herein is defined as a chain of
nucleotides. Furthermore, nucleic acids are polymers of nucleotides.
Thus, nucleic acids and polynucleotides as used herein are
interchangeable. One skilled in the art has the general knowledge that
nucleic acids are polynucleotides, which can be hydrolyzed into the
monomeric "nucleotides." The monomeric nucleotides can be hydrolyzed into
nucleosides. As used herein polynucleotides include, but are not limited
to, all nucleic acid sequences which are obtained by any means available
in the art, including, without limitation, recombinant means, i.e., the
cloning of nucleic acid sequences from a recombinant library or a cell
genome, using ordinary cloning technology and PCR.TM., and the like, and
by synthetic means.

[0074] As used herein, the term "alkyl," by itself or as part of another
substituent means, unless otherwise stated, a straight or branched chain
hydrocarbon having the number of carbon atoms designated (i.e.
C.sub.1-.sub.6 means one to six carbon atoms) and includes straight,
branched chain, or cyclic substituent groups. Examples include methyl,
ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl,
hexyl, and cyclopropylmethyl. Most preferred is (C.sub.1-C.sub.6)alkyl,
particularly ethyl, methyl, isopropyl, isobutyl, n-pentyl, n-hexyl and
cyclopropylmethyl.

[0075] As used herein, the term "substituted alkyl" means alkyl, as
defined above, substituted by one, two or three substituents selected
from the group consisting of halogen, --OH, alkoxy, --NH.sub.2,
--N(CH.sub.3).sub.2, --C(.dbd.O)OH, trifluoromethyl, --C.ident.N,
--C(.dbd.O)O(C.sub.1-C.sub.4)alkyl, --C(.dbd.O)NH.sub.2,
--SO.sub.2NH.sub.2, --C(.dbd.NH)NH.sub.2, and --NO.sub.2, preferably
containing one or two substituents selected from halogen, --OH, alkoxy,
--NH.sub.2, trifluoromethyl, --N(CH.sub.3).sub.2, and --C(.dbd.O)OH, more
preferably selected from halogen, alkoxy and --OH. Examples of
substituted alkyls include, but are not limited to, 2,2-difluoropropyl,
2-carboxycyclopentyl and 3-chloropropyl.

[0076] As used herein, the term "alkoxy" employed alone or in combination
with other terms means, unless otherwise stated, an alkyl group having
the designated number of carbon atoms, as defined above, connected to the
rest of the molecule via an oxygen atom, such as, for example, methoxy,
ethoxy, 1-propoxy, 2-propoxy (isopropoxy) and the higher homologs and
isomers. Preferred are (C.sub.1-C.sub.3) alkoxy, particularly ethoxy and
methoxy.

[0077] As used herein, the term "halo" or "halogen" alone or as part of
another substituent means, unless otherwise stated, a fluorine, chlorine,
bromine, or iodine atom, preferably, fluorine, chlorine, or bromine, more
preferably, fluorine or chlorine.

[0078] As used herein, the term "heteroalkyl" by itself or in combination
with another term means, unless otherwise stated, a stable straight or
branched chain alkyl group consisting of the stated number of carbon
atoms and one or two heteroatoms selected from the group consisting of O,
N, and S, and wherein the nitrogen and sulfur atoms may be optionally
oxidized and the nitrogen heteroatom may be optionally quaternized. The
heteroatom(s) may be placed at any position of the heteroalkyl group,
including between the rest of the heteroalkyl group and the fragment to
which it is attached, as well as attached to the most distal carbon atom
in the heteroalkyl group. Examples include:
--O--CH.sub.2--CH.sub.2--CH.sub.3, --CH.sub.2--CH.sub.2--CH.sub.2--OH,
--CH.sub.2--CH.sub.2--NH--CH.sub.3, --CH.sub.2--S--CH.sub.2--CH.sub.3,
and --CH.sub.2CH.sub.2--S(.dbd.O)--CH.sub.3. Up to two heteroatoms may be
consecutive, such as, for example, --CH.sub.2--NH--OCH.sub.3, or
--CH.sub.2--CH.sub.2--S--S--CH.sub.3

[0079] As used herein, the term "aromatic" refers to a carbocycle or
heterocycle with one or more polyunsaturated rings and having aromatic
character, i.e. having (4n+2) delocalized .pi. (pi) electrons, where n is
an integer.

[0080] As used herein, the term "aryl," employed alone or in combination
with other terms, means, unless otherwise stated, a carbocyclic aromatic
system containing one or more rings (typically one, two or three rings)
wherein such rings may be attached together in a pendent manner, such as
a biphenyl, or may be fused, such as naphthalene. Examples include
phenyl, anthracyl, and naphthyl. Preferred are phenyl and naphthyl, most
preferred is phenyl.

[0081] As used herein, the term "aryl-(C.sub.1-C.sub.3)alkyl" means a
functional group wherein a one to three carbon alkylene chain is attached
to an aryl group, e.g., --CH.sub.2CH.sub.2-phenyl. Preferred is
aryl-CH.sub.2-- and aryl-CH(CH.sub.3)--. The term "substituted
aryl-(C.sub.1-C.sub.3)alkyl" means an aryl-(C.sub.1-C.sub.3)alkyl
functional group in which the aryl group is substituted. Preferred is
substituted aryl(CH.sub.2)--. Similarly, the term
"heteroaryl-(C.sub.1-C.sub.3)alkyl" means a functional group wherein a
one to three carbon alkylene chain is attached to a heteroaryl group,
e.g., --CH.sub.2CH.sub.2-pyridyl. Preferred is heteroaryl-(CH.sub.2)--.
The term "substituted heteroaryl-(C.sub.1-C.sub.3)alkyl" means a
heteroaryl-(C.sub.1-C.sub.3)alkyl functional group in which the
heteroaryl group is substituted. Preferred is substituted
heteroaryl-(CH.sub.2)--.

[0082] As used herein, the term "heterocycle" or "heterocyclyl" or
"heterocyclic" by itself or as part of another substituent means, unless
otherwise stated, an unsubstituted or substituted, stable, mono- or
multi-cyclic heterocyclic ring system that consists of carbon atoms and
at least one heteroatom selected from the group consisting of N, O, and
S, and wherein the nitrogen and sulfur heteroatoms may be optionally
oxidized, and the nitrogen atom may be optionally quaternized. The
heterocyclic system may be attached, unless otherwise stated, at any
heteroatom or carbon atom that affords a stable structure. A heterocycle
may be aromatic or non-aromatic in nature. In one embodiment, the
heterocycle is a heteroaryl.

[0083] As used herein, the term "heteroaryl" or "heteroaromatic" refers to
a heterocycle having aromatic character. A polycyclic heteroaryl may
include one or more rings that are partially saturated. Examples include
tetrahydroquinoline and 2,3-dihydrobenzofuryl.

[0087] The aforementioned listing of heterocyclyl and heteroaryl moieties
is intended to be representative and not limiting.

[0088] As used herein, the term "substituted" means that an atom or group
of atoms has replaced hydrogen as the substituent attached to another
group.

[0089] For aryl, aryl-(C.sub.1-C.sub.3)alkyl and heterocyclyl groups, the
term "substituted" as applied to the rings of these groups refers to any
level of substitution, namely mono-, di-, tri-, tetra-, or
penta-substitution, where such substitution is permitted. The
substituents are independently selected, and substitution may be at any
chemically accessible position. In one embodiment, the substituents vary
in number between one and four. In another embodiment, the substituents
vary in number between one and three. In yet another embodiment, the
substituents vary in number between one and two. In yet another
embodiment, the substituents are independently selected from the group
consisting of C.sub.1-6 alkyl, --OH, C.sub.1-6 alkoxy, halo, amino,
acetamido and nitro. In yet another embodiment, the substituents are
independently selected from the group consisting of C.sub.1-6 alkyl,
C.sub.1-6 alkoxy, halo, acetamido, and nitro. As used herein, where a
substituent is an alkyl or alkoxy group, the carbon chain may be
branched, straight or cyclic, with straight being preferred.

[0090] By the term "specifically binds," as used herein, is meant a
molecule, such as an antibody or a small molecule, which recognizes and
binds to another molecule or feature, but does not substantially
recognize or bind other molecules or features in a sample.

[0091] The phrase "inhibit," as used herein, means to reduce a molecule, a
reaction, an interaction, a gene, an mRNA, and/or a protein's expression,
stability, function or activity by a measurable amount or to prevent
entirely. Inhibitors are compounds that, e.g., bind to, partially or
totally block stimulation, decrease, prevent, delay activation,
inactivate, desensitize, or down regulate a protein, a gene, and an mRNA
stability, expression, function and activity, e.g., antagonists.

[0092] "Effective amount" or "therapeutically effective amount" are used
interchangeably herein, and refer to an amount of a compound,
formulation, material, or composition, as described herein effective to
achieve a particular biological result. Such results may include, but are
not limited to, the treatment of a disease or condition as determined by
any means suitable in the art.

[0093] As used herein, the term "pharmaceutical composition" refers to a
mixture of at least one compound of the invention with other chemical
components, such as carriers, stabilizers, diluents, dispersing agents,
suspending agents, thickening agents, and/or excipients. The
pharmaceutical composition facilitates administration of the compound to
an organism. Multiple techniques of administering a compound exist in the
art including, but not limited to, intravenous, oral, aerosol,
parenteral, ophthalmic, pulmonary and topical administration.

[0094] "Pharmaceutically acceptable" refers to those properties and/or
substances which are acceptable to the patient from a
pharmacological/toxicological point of view and to the manufacturing
pharmaceutical chemist from a physical/chemical point of view regarding
composition, formulation, stability, patient acceptance and
bioavailability. "Pharmaceutically acceptable carrier" refers to a medium
that does not interfere with the effectiveness of the biological activity
of the active ingredient(s) and is not toxic to the host to which it is
administered.

[0095] As used herein, the term "pharmaceutically acceptable carrier"
means a pharmaceutically acceptable material, composition or carrier,
such as a liquid or solid filler, stabilizer, dispersing agent,
suspending agent, diluent, excipient, thickening agent, solvent or
encapsulating material, involved in carrying or transporting a compound
useful within the invention within or to the patient such that it may
perform its intended function. Typically, such constructs are carried or
transported from one organ, or portion of the body, to another organ, or
portion of the body. Each carrier must be "acceptable" in the sense of
being compatible with the other ingredients of the formulation, including
the compound useful within the invention, and not injurious to the
patient. Some examples of materials that may serve as pharmaceutically
acceptable carriers include: sugars, such as lactose, glucose and
sucrose; starches, such as corn starch and potato starch; cellulose, and
its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose
and cellulose acetate; powdered tragacanth; malt; gelatin; talc;
excipients, such as cocoa butter and suppository waxes; oils, such as
peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn
oil and soybean oil; glycols, such as propylene glycol; polyols, such as
glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as
ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium
hydroxide and aluminum hydroxide; surface active agents; alginic acid;
pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;
phosphate buffer solutions; and other non-toxic compatible substances
employed in pharmaceutical formulations. As used herein,
"pharmaceutically acceptable carrier" also includes any and all coatings,
antibacterial and antifungal agents, and absorption delaying agents, and
the like that are compatible with the activity of the compound useful
within the invention, and are physiologically acceptable to the patient.
Supplementary active compounds may also be incorporated into the
compositions. The "pharmaceutically acceptable carrier" may further
include a pharmaceutically acceptable salt of the compound useful within
the invention. Other additional ingredients that may be included in the
pharmaceutical compositions used in the practice of the invention are
known in the art and described, for example in Remington's Pharmaceutical
Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, Pa.), which is
incorporated herein by reference.

[0097] An "individual", "patient" or "subject", as that term is used
herein, includes a member of any animal species including, but are not
limited to, birds, humans and other primates, and other mammals including
commercially relevant mammals such as cattle, pigs, horses, sheep, cats,
and dogs. Preferably, the subject is a human.

[0098] The term "treat" or "treating", as used herein, means reducing the
frequency with which symptoms are experienced by a subject or
administering an agent or compound to reduce the frequency and/or
severity with which symptoms are experienced. As used herein, "alleviate"
is used interchangeably with the term "treat." Treating a disease,
disorder or condition may or may not include complete eradication or
elimination of the symptom. The term "therapeutic" as used herein means a
treatment and/or prophylaxis. A therapeutic effect is obtained by
suppression, remission, or eradication of UVR-induced skin damage.

[0099] As used herein, the term "container" includes any receptacle for
holding the pharmaceutical composition. For example, in one embodiment,
the container is the packaging that contains the pharmaceutical
composition. In other embodiments, the container is not the packaging
that contains the pharmaceutical composition, i.e., the container is a
receptacle, such as a box or vial that contains the packaged
pharmaceutical composition or unpackaged pharmaceutical composition and
the instructions for use of the pharmaceutical composition. Moreover,
packaging techniques are well known in the art. It should be understood
that the instructions for use of the pharmaceutical composition may be
contained on the packaging containing the pharmaceutical composition, and
as such the instructions form an increased functional relationship to the
packaged product. However, it should be understood that the instructions
may contain information pertaining to the compound's ability to perform
its intended function, e.g., treating or preventing a disease in a
subject.

[0100] "Instructional material," as that term is used herein, includes a
publication, a recording, a diagram, or any other medium of expression
which can be used to communicate the usefulness of the composition and/or
compound of the invention in a kit. The instructional material of the kit
may, for example, be affixed to a container that contains the compound
and/or composition of the invention or be shipped together with a
container which contains the compound and/or composition. Alternatively,
the instructional material may be shipped separately from the container
with the intention that the recipient uses the instructional material and
the compound cooperatively. Delivery of the instructional material may
be, for example, by physical delivery of the publication or other medium
of expression communicating the usefulness of the kit, or may
alternatively be achieved by electronic transmission, for example by
means of a computer, such as by electronic mail, or download from a
website.

[0101] Throughout this disclosure, various aspects of the invention can be
presented in a range format. It should be understood that the description
in range format is merely for convenience and brevity and should not be
construed as an inflexible limitation on the scope of the invention.
Accordingly, the description of a range should be considered to have
specifically disclosed all the possible sub-ranges as well as individual
numerical values within that range. For example, description of a range
such as from 1 to 6 should be considered to have specifically disclosed
sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4,
from 2 to 6, from 3 to 6 etc., as well as individual numbers within that
range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies
regardless of the breadth of the range.

Compounds Useful Within the Invention

[0102] In one aspect, the compound useful within the compositions and
methods of the invention is the compound of Formula (1):

[0107] In one embodiment, R.sup.3 is H, C.sub.1-C.sub.6 alkyl,
O(C.sub.1-C.sub.6 alkyl), F or Cl. In another embodiment, R.sup.3 is H,
C.sub.1-C.sub.6 alkyl, or O(C.sub.1-C.sub.6 alkyl). In yet another
embodiment, R.sup.3 is H, methyl, ethyl, methoxy or ethoxy. In yet
another embodiment, R.sup.3 is H.

[0108] In one embodiment, the compound of Formula (1) is selected from the
group consisting of:

[0112] In another aspect, the compound useful within the compositions and
methods of the invention is salazinic acid, also known as
1,4,10-trihydroxy-5-(hydroxymethyl)-8-methyl-3,7-dioxo-3,7-dihydro-1H-ben-
zo[6,7][1,4]dioxepino[2,3-e]isobenzofuran-11-carbaldehyde, which has
formula (2):

##STR00011##

or a salt thereof.

[0113] In yet another aspect, the compound useful within the compositions
and methods of the invention is stictic acid, also known as scopularic
acid or 1,4-dihydroxy-10-methoxy-5,8-dimethyl-3,7-dioxo-3,7-dihydro-1H-be-
nzo[6,7][1,4]dioxepino[2,3-e]isobenzofuran-11-carbaldehyde, which has
formula (3):

##STR00012##

or a salt thereof.

[0114] Compounds useful within the methods of the invention may be
synthesized using techniques well-known in the art of organic synthesis
or obtained from commercial sources.

Compositions of the Invention

[0115] In one aspect, the invention includes a pharmaceutical composition
comprising a compound of Formula (1) and a pharmaceutically acceptable
carrier. In one embodiment, the pharmaceutical composition further
comprises a chemotherapeutic agent. In another embodiment, the agent is
selected from the group consisting of an alkylating agent,
antimetabolite, anthracycline, plant alkaloid, plant terpenoid,
topoisomerase inhibitor, and antineoplastic.

[0116] In another aspect, the invention includes a pharmaceutical
composition comprising a compound of Formula (2), a chemotherapeutic
agent, and a pharmaceutically acceptable carrier.

[0117] In another aspect, the invention includes a pharmaceutical
composition comprising a compound of Formula (3), a chemotherapeutic
agent, and a pharmaceutically acceptable carrier.

Salts of the Compounds of the Invention

[0118] The compounds described herein may form salts with acids or bases,
and such salts are included in the present invention. In one embodiment,
the salts are pharmaceutically acceptable salts. The term "salts"
embraces addition salts of free acids or free bases that are compounds of
the invention. The term "pharmaceutically acceptable salt" refers to
salts that possess toxicity profiles within a range that affords utility
in pharmaceutical applications. Pharmaceutically unacceptable salts may
nonetheless possess properties such as high crystallinity, which have
utility in the practice of the present invention, such as for example
utility in process of synthesis, purification or formulation of compounds
of the invention.

[0121] Suitable pharmaceutically acceptable base addition salts of
compounds of the invention include, for example, metallic salts including
alkali metal, alkaline earth metal and transition metal salts such as,
for example, calcium, magnesium, potassium, sodium and zinc salts.
Pharmaceutically acceptable base addition salts also include organic
salts made from basic amines such as, for example,
N,N'-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine,
ethylenediamine, meglumine (N-methylglucamine) and procaine. Examples of
pharmaceutically unacceptable base addition salts include lithium salts
and cyanate salts. All of these salts may be prepared from the
corresponding compound by reacting, for example, the appropriate acid or
base

Methods of the Invention

[0122] The invention includes a method of treating or preventing cancer in
a subject in need thereof. The method comprises the step of administering
to the subject a pharmaceutical composition comprising a pharmaceutically
acceptable carrier and a pharmaceutically effective amount of a compound
of Formula (1):

[0124] R.sup.3 is H, C.sub.1-C.sub.6 alkyl, O(C.sub.1-C.sub.6 alkyl), F,
Cl, Br or I; or a salt thereof. The method further comprises the step of
administering to the subject a treatment selected from the group
consisting of (i) radiation therapy, and (ii) a pharmaceutical
composition comprising a pharmaceutically effective amount of a
chemotherapeutic agent, whereby treating or preventing the cancer in the
subject.

[0125] In one embodiment, the compound of Formula (1) is selected from the
group consisting of
(E)-3-benzyl-2-(2-(pyridin-3-yl)vinyl)quinazolin-4(3H)-one (1a),
(E)-3-ethyl-2-(2-(pyridin-3-yl)vinyl)quinazolin-4(3H)-one (1b),
(E)-2-(2-(pyridin-3-yl)vinyl)-3-(m-tolyl)quinazolin-4(3H)-one (1c),
mixtures thereof and salts thereof.

[0126] In one embodiment, administering of the compound of Formula (1) is
performed at least 24 hours prior to administering to the subject the
radiation therapy or the chemotherapeutic agent. In another embodiment,
administering of the compound of Formula (1) is performed at least 12
hours prior to administering to the subject the radiation therapy or the
chemotherapeutic agent. In yet another embodiment, administering of the
compound of Formula (1) is performed at least 6 hours prior to
administering to the subject the radiation therapy or the
chemotherapeutic agent. In yet another embodiment, administering of the
compound of Formula (1) is performed at least 3 hours prior to
administering to the subject the radiation therapy or the
chemotherapeutic agent. In yet another embodiment, administering of the
compound of Formula (1) is performed at least 1 hour prior to
administering to the subject the radiation therapy or the
chemotherapeutic agent. In yet another embodiment, the composition
comprising the compound of Formula (1) is co-administered to the subject
with the radiation therapy or the composition comprising the
chemotherapeutic agent. In yet another embodiment, the compound of
Formula (1) and the chemotherapeutic agent are co-formulated in a
pharmaceutical composition. In yet another embodiment, the subject is a
human.

[0127] The invention also includes a method of treating or preventing
cancer in a subject in need thereof. The method comprises the step of
administering to the subject a pharmaceutical composition comprising a
pharmaceutically acceptable carrier and a pharmaceutically effective
amount of a compound of Formula (2):

##STR00014##

or a salt thereof. The method further comprises the step of administering
to the subject a treatment selected from the group consisting of (i)
radiation therapy, and (ii) a pharmaceutical composition comprising a
pharmaceutically effective amount of a chemotherapeutic agent; whereby
treating or preventing the cancer in the subject.

[0128] In one embodiment, administering of the compound of Formula (2) is
performed at least 24 hours prior to administering to the subject the
radiation therapy or the chemotherapeutic agent. In another embodiment,
administering of the compound of Formula (2) is performed at least 12
hours prior to administering to the subject the radiation therapy or the
chemotherapeutic agent. In yet another embodiment, administering of the
compound of Formula (2) is performed at least 6 hours prior to
administering to the subject the radiation therapy or the
chemotherapeutic agent. In yet another embodiment, administering of the
compound of Formula (2) is performed at least 3 hours prior to
administering to the subject the radiation therapy or the
chemotherapeutic agent. In yet another embodiment, administering of the
compound of Formula (2) is performed at least 1 hour prior to
administering to the subject the radiation therapy or the
chemotherapeutic agent. In yet another embodiment, the composition
comprising the compound of Formula (2) is co-administered to the subject
with the radiation therapy or the composition comprising the
chemotherapeutic agent. In yet another embodiment, the compound of
Formula (2) and the chemotherapeutic agent are co-formulated in a
pharmaceutical composition. In yet another embodiment, the subject is a
human.

[0129] The invention further includes a method of treating or preventing
cancer in a subject in need thereof. The method comprises the step of
administering to the subject a pharmaceutical composition comprising a
pharmaceutically acceptable carrier and a pharmaceutically effective
amount of a compound of Formula (3):

##STR00015##

or a salt thereof. The method further comprises the step of administering
to the subject a treatment selected from the group consisting of (i)
radiation therapy, and (ii) a pharmaceutical composition comprising a
pharmaceutically effective amount of a chemotherapeutic agent; whereby
treating or preventing the cancer in the subject.

[0130] In one embodiment, administering of the compound of Formula (3) is
performed at least 24 hours prior to administering to the subject the
radiation therapy or the chemotherapeutic agent. In another embodiment,
administering of the compound of Formula (3) is performed at least 12
hours prior to administering to the subject the radiation therapy or the
chemotherapeutic agent. In yet another embodiment, administering of the
compound of Formula (3) is performed at least 6 hours prior to
administering to the subject the radiation therapy or the
chemotherapeutic agent. In yet another embodiment, administering of the
compound of Formula (3) is performed at least 3 hours prior to
administering to the subject the radiation therapy or the
chemotherapeutic agent. In yet another embodiment, administering of the
compound of Formula (3) is performed at least 1 hour prior to
administering to the subject the radiation therapy or the
chemotherapeutic agent. In yet another embodiment, the composition
comprising the compound of Formula (3) is co-administered to the subject
with the radiation therapy or the composition comprising the
chemotherapeutic agent. In yet another embodiment, the compound of
Formula (3) and the chemotherapeutic agent are co-formulated in a
pharmaceutical composition. In yet another embodiment, the subject is a
human.

Combination Therapies

[0131] The compounds contemplated within the invention or salts thereof
may be useful in the methods of present invention in combination with
radiation therapy and/or a compound useful for treating cancer (generally
referred to as "chemotherapeutic agent"). These additional compounds may
comprise compounds of the present invention or compounds (such as
commercially available compounds) known to treat, prevent, or reduce the
symptoms of cancer. In one embodiment, the combination of a compound
contemplated within the invention and a chemotherapeutic agent has
additive, complementary or synergistic effects in the treatment of cancer
in a subject, or prevention of cancer in a subject. In another
embodiment, the combination of a compound contemplated within the
invention and radiation therapy has additive, complementary or
synergistic effects in the treatment of cancer in a subject, or
prevention of cancer in a subject.

Radiation Therapy

[0132] In one aspect, a compound contemplated within the invention or a
salt thereof may be used in combination with radiation therapy.

[0133] Radiation therapy, radiation oncology, or radiotherapy, sometimes
abbreviated to XRT, is the medical use of ionizing radiation as part of
cancer treatment to control malignant cells. Radiotherapy may be used for
curative or adjuvant treatment. It is used as palliative treatment (where
cure is not possible and the aim is for local disease control or
symptomatic relief) or as therapeutic treatment (where the therapy has
survival benefit and it can be curative).

[0134] Radiotherapy is used for the treatment of malignant cancer, and may
be used as a primary or adjuvant modality. It is also common to combine
radiotherapy with surgery, chemotherapy, hormone therapy, immunotherapy
or a mixture of the four. Most common cancer types can be treated with
radiotherapy in some way. The precise treatment intent (curative,
adjuvant, neoadjuvant, therapeutic, or palliative) will depend on the
tumor type, location, and stage, as well as the general health of the
patient.

[0135] Radiation therapy is commonly applied to the cancerous tumor. The
radiation fields may also include the draining lymph nodes if they are
clinically or radiologically involved with tumor, or if there is thought
to be a risk of subclinical malignant spread. Brachytherapy, in which a
radiation source is placed inside or next to the area requiring
treatment, is another form of radiation therapy that minimizes exposure
to healthy tissue during procedures to treat cancers of the breast,
prostate and other organs.

[0136] The amount of radiation used in photon radiation therapy is
measured in gray (Gy), and varies depending on the type and stage of
cancer being treated. For curative cases, the typical dose for a solid
epithelial tumor ranges from 60 to 80 Gy, while lymphomas are treated
with 20 to 40 Gy. Preventative (adjuvant) doses are typically around
45-60 Gy in 1.8-2 Gy fractions (for breast, head, and neck cancers.) Many
other factors are considered by radiation oncologists when selecting a
dose, including whether the patient is receiving chemotherapy, patient
comorbidities, whether radiation therapy is being administered before or
after surgery, and the degree of success of surgery.

Chemotherapeutic Agents

[0137] In one aspect of the invention, a compound of the invention or a
salt thereof may be used in combination with a chemotherapeutic agent.

[0138] In one embodiment, a compound of the invention is co-administered
with a chemotherapeutic agent to the subject in need thereof. In another
embodiment, a compound of the invention and a chemotherapeutic agent are
administered to the subject as part of the same pharmaceutical
formulation. In yet another embodiment, a compound of the invention and a
chemotherapeutic agent are administered separately to the subject in need
thereof.

[0139] Most of the approved chemotherapeutic agents may be divided into
alkylating agents, antimetabolites, anthracyclines, plant alkaloids and
terpenoids, topoisomerase inhibitors, antineoplastics and other
antitumour
agents.http//en.wikipedia.org/wiki/Chemotherapy-cite_note-takimoto-7
These drugs affect cell division or DNA synthesis and function directly
or indirectly.

[0140] Some newer chemotherapeutic agents do not directly interfere with
DNA synthesis and function. These include monoclonal antibodies and
tyrosine kinase inhibitors e.g. imatinib mesylate (Gleevec or Glivec),
which directly targets a molecular abnormality in certain types of cancer
(chronic myelogenous leukemia, gastrointestinal stromal tumors), and are
generally referred to as targeted therapies.

[0141] In addition, some drugs that modulate tumor cell behavior without
directly attacking those cells, such as hormones, may be used in the
treatment of cancer.

[0144] Anti-metabolites masquerade as purines (azathioprine,
mercaptopurine) or pyrimidines, which are building blocks of DNA. By
competing out naturally occurring purines or pyrimidines,
anti-metabolites prevent these building blocks from becoming incorporated
into DNA during the "S" phase (of the cell cycle), thus stopping normal
development and division. Anti-metabolites also affect RNA synthesis. Due
to their efficiency, anti-metabolites are the most widely used
cytostatics.

[0147] Vinca alkaloids bind to specific sites on tubulin, inhibiting
assembly of tubulin into microtubules (M phase of the cell cycle). The
vinca alkaloids include vincristine, vinblastine, vinorelbine and
vindesine.

[0148] (b) Podophyllotoxin

[0149] Podophyllotoxin is a plant-derived compound said to help with
digestion and used to produce two other cytostatic drugs, etoposide and
teniposide. They prevent the cell from entering the G1 phase (the start
of DNA replication) and the replication of DNA (the S phase). The exact
mechanism of its action is unknown. The substance has been primarily
obtained from the American Mayapple (Podophyllum peltatum). Recently it
has been discovered that a rare Himalayan Mayapple (Podophyllum
hexandrum) contains it in a much greater quantity, but, as the plant is
endangered, its supply is limited. Studies have been conducted to isolate
the genes involved in the substance's production, so that it could be
obtained recombinantly.

[0150] (c) Taxanes

[0151] The prototype taxane is the natural product paclitaxel, originally
known as Taxol and first derived from the bark of the Pacific Yew tree.
Docetaxel is a semi-synthetic analogue of paclitaxel. Taxanes enhance
stability of microtubules, preventing the separation of chromosomes
during anaphase.

Topoisomerase Inhibitors:

[0152] Topoisomerases are essential enzymes that maintain the topology of
DNA. Inhibition of type I or type II topoisomerases interferes with both
transcription and replication of DNA by upsetting proper DNA
supercoiling. Type I topoisomerase inhibitors include camptothecins:
irinotecan and topotecan. Type II inhibitors include amsacrine,
etoposide, etoposide phosphate, and teniposide. These are semisynthetic
derivatives of epipodophyllotoxins, alkaloids naturally occurring in the
root of American Mayapple (Podophyllum peltatum).

Antineoplastics:

[0153] These include the immunosuppressant dactinomycin (which is used in
kidney transplantations), doxorubicin, epirubicin, bleomycin and others.

[0154] Anticancer agents working through different cytotoxic mechanisms
may also be combined in "chemotherapy regimens" in order to target a
specific type of cancer. Chemotherapy regimens are often identified by
acronyms, identifying the agents used in combination. However, the
letters used are not consistent across regimens, and in some cases (for
example, "BEACOPP"), the same letter combination is used to represent two
different treatments. Non-limiting examples of combinations used in
clinical settings are listed below, in terms of acronyms, compositions
and cancer types:

[0194] A synergistic effect may be calculated, for example, using suitable
methods such as, for example, the Sigmoid-E.sub.max equation (Holford &
Scheiner, 19981, Clin. Pharmacokinet. 6: 429-453), the equation of Loewe
additivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114:
313-326) and the median-effect equation (Chou & Talalay, 1984, Adv.
Enzyme Regul. 22: 27-55). Each equation referred to above may be applied
to experimental data to generate a corresponding graph to aid in
assessing the effects of the drug combination. The corresponding graphs
associated with the equations referred to above are the
concentration-effect curve, isobologram curve and combination index
curve, respectively.

Pharmaceutical Compositions and Therapies

[0195] Administration of a compound useful within the invention may be
achieved in a number of different ways, using methods known in the art.
The therapeutic and prophylactic methods of the invention thus encompass
the use of pharmaceutical compositions comprising the compounds useful
within the invention to practice the methods of the invention. The
pharmaceutical compositions useful for practicing the invention may be
administered to deliver a dose of 1 ng/kg/day to 100 mg/kg/day.

[0196] The relative amounts of the active ingredient, the pharmaceutically
acceptable carrier, and any additional ingredients in a pharmaceutical
composition of the invention will vary, depending upon the identity,
size, and condition of the subject treated and further depending upon the
route by which the composition is to be administered. By way of example,
the composition may comprise between 0.1% and 100% (w/w) active
ingredient.

[0197] Although the description of pharmaceutical compositions provided
herein are principally directed to pharmaceutical compositions that are
suitable for ethical administration to humans, it will be understood by
the skilled artisan that such compositions are generally suitable for
administration to animals of all sorts. Modification of pharmaceutical
compositions suitable for administration to humans in order to render the
compositions suitable for administration to various animals is well
understood, and the ordinarily skilled veterinary pharmacologist can
design and perform such modification with merely ordinary, if any,
experimentation. Subjects to which administration of the pharmaceutical
compositions of the invention is contemplated include, but are not
limited to, humans and other primates, mammals including commercially
relevant mammals such as non-human primates, cattle, pigs, horses, sheep,
cats, and dogs.

[0198] Typically, dosages which may be administered in a method of the
invention to an animal, preferably a human, range in amount from 0.5
.mu.g to about 50 mg per kilogram of body weight of the animal. While the
precise dosage administered will vary depending upon any number of
factors, including but not limited to, the type of animal and type of
disease state being treated, the age of the animal and the route of
administration, the dosage of the compound will preferably vary from
about 1 .mu.g to about 10 mg per kilogram of body weight of the animal.
More preferably, the dosage will vary from about 3 .mu.g to about 1 mg
per kilogram of body weight of the animal.

[0199] Pharmaceutical compositions that are useful in the methods of the
invention may be prepared, packaged, or sold in formulations suitable for
oral, parenteral, topical, buccal, or another route of administration.
Other contemplated formulations include projected nanoparticles,
liposomal preparations, resealed erythrocytes containing the active
ingredient, and immunologically-based formulations.

[0200] The formulations of the pharmaceutical compositions described
herein may be prepared by any method known or hereafter developed in the
art of pharmacology. In general, such preparatory methods include the
step of bringing the active ingredient into association with a
pharmaceutically acceptable carrier or one or more other accessory
ingredients, and then, if necessary or desirable, shaping or packaging
the product into a desired single- or multi-dose unit.

[0201] A pharmaceutical composition of the invention may be prepared,
packaged, or sold in bulk, as a single unit dose, or as a plurality of
single unit doses. As used herein, a "unit dose" is discrete amount of
the pharmaceutical composition comprising a predetermined amount of the
active ingredient. The amount of the active ingredient is generally equal
to the dosage of the active ingredient that would be administered to a
subject or a convenient fraction of such a dosage such as, for example,
one-half or one-third of such a dosage.

[0202] In one embodiment, the compositions of the invention are formulated
using one or more pharmaceutically acceptable excipients or carriers. In
one embodiment, the pharmaceutical compositions of the invention comprise
a therapeutically effective amount of a compound or conjugate of the
invention and a pharmaceutically acceptable carrier. Pharmaceutically
acceptable carriers that are useful, include, but are not limited to,
glycerol, water, saline, ethanol and other pharmaceutically acceptable
salt solutions such as phosphates and salts of organic acids. Examples of
these and other pharmaceutically acceptable carriers are described in
Remington's Pharmaceutical Sciences (1991, Mack Publication Co., N.J.).

[0203] The carrier may be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene glycol,
and liquid polyethylene glycol, and the like), suitable mixtures thereof,
and vegetable oils. The proper fluidity may be maintained, for example,
by the use of a coating such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by the use of
surfactants. Prevention of the action of microorganisms may be achieved
by various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many
cases, it will be preferable to include isotonic agents, for example,
sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol,
in the composition. Prolonged absorption of the injectable compositions
may be brought about by including in the composition an agent that delays
absorption, for example, aluminum monostearate or gelatin. In one
embodiment, the pharmaceutically acceptable carrier is not DMSO alone.

[0204] Formulations may be employed in admixtures with conventional
excipients, i.e., pharmaceutically acceptable organic or inorganic
carrier substances suitable for oral, parenteral, nasal, intravenous,
subcutaneous, enteral, or any other suitable mode of administration,
known to the art. The pharmaceutical preparations may be sterilized and
if desired mixed with auxiliary agents, e.g., lubricants, preservatives,
stabilizers, wetting agents, emulsifiers, salts for influencing osmotic
pressure buffers, coloring, flavoring and/or aromatic substances and the
like. They may also be combined where desired with other active agents,
e.g., other analgesic agents.

[0206] The composition of the invention may comprise a preservative from
about 0.005% to 2.0% by total weight of the composition. The preservative
is used to prevent spoilage in the case of exposure to contaminants in
the environment. Examples of preservatives useful in accordance with the
invention included but are not limited to those selected from the group
consisting of benzyl alcohol, sorbic acid, parabens, imidurea and
combinations thereof. A particularly preferred preservative is a
combination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic
acid.

[0207] The composition preferably includes an anti-oxidant and a chelating
agent that inhibits the degradation of the compound. Preferred
antioxidants for some compounds are BHT, BHA, alpha-tocopherol and
ascorbic acid in the preferred range of about 0.01% to 0.3% and more
preferably BHT in the range of 0.03% to 0.1% by weight by total weight of
the composition. Preferably, the chelating agent is present in an amount
of from 0.01% to 0.5% by weight by total weight of the composition.
Particularly preferred chelating agents include edetate salts (e.g.
disodium edetate) and citric acid in the weight range of about 0.01% to
0.20% and more preferably in the range of 0.02% to 0.10% by weight by
total weight of the composition. The chelating agent is useful for
chelating metal ions in the composition that may be detrimental to the
shelf life of the formulation. While BHT and disodium edetate are the
particularly preferred antioxidant and chelating agent respectively for
some compounds, other suitable and equivalent antioxidants and chelating
agents may be substituted therefore as would be known to those skilled in
the art.

[0208] Liquid suspensions may be prepared using conventional methods to
achieve suspension of the active ingredient in an aqueous or oily
vehicle. Aqueous vehicles include, for example, water, and isotonic
saline. Oily vehicles include, for example, almond oil, oily esters,
ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut
oil, fractionated vegetable oils, and mineral oils such as liquid
paraffin. Liquid suspensions may further comprise one or more additional
ingredients including, but not limited to, suspending agents, dispersing
or wetting agents, emulsifying agents, demulcents, preservatives,
buffers, salts, flavorings, coloring agents, and sweetening agents. Oily
suspensions may further comprise a thickening agent. Known suspending
agents include, but are not limited to, sorbitol syrup, hydrogenated
edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum
acacia, and cellulose derivatives such as sodium carboxymethylcellulose,
methylcellulose, hydroxypropylmethylcellulose. Known dispersing or
wetting agents include, but are not limited to, naturally-occurring
phosphatides such as lecithin, condensation products of an alkylene oxide
with a fatty acid, with a long chain aliphatic alcohol, with a partial
ester derived from a fatty acid and a hexitol, or with a partial ester
derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene
stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol
monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known
emulsifying agents include, but are not limited to, lecithin, and acacia.
Known preservatives include, but are not limited to, methyl, ethyl, or
n-propyl-para-hydroxybenzoates, ascorbic acid, and sorbic acid. Known
sweetening agents include, for example, glycerol, propylene glycol,
sorbitol, sucrose, and saccharin. Known thickening agents for oily
suspensions include, for example, beeswax, hard paraffin, and cetyl
alcohol.

[0209] Liquid solutions of the active ingredient in aqueous or oily
solvents may be prepared in substantially the same manner as liquid
suspensions, the primary difference being that the active ingredient is
dissolved, rather than suspended in the solvent. As used herein, an
"oily" liquid is one which comprises a carbon-containing liquid molecule
and which exhibits a less polar character than water. Liquid solutions of
the pharmaceutical composition of the invention may comprise each of the
components described with regard to liquid suspensions, it being
understood that suspending agents will not necessarily aid dissolution of
the active ingredient in the solvent. Aqueous solvents include, for
example, water, and isotonic saline. Oily solvents include, for example,
almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis,
olive, sesame, or coconut oil, fractionated vegetable oils, and mineral
oils such as liquid paraffin.

[0210] Powdered and granular formulations of a pharmaceutical preparation
of the invention may be prepared using known methods. Such formulations
may be administered directly to a subject, used, for example, to form
tablets, to fill capsules, or to prepare an aqueous or oily suspension or
solution by addition of an aqueous or oily vehicle thereto. Each of these
formulations may further comprise one or more of dispersing or wetting
agent, a suspending agent, and a preservative. Additional excipients,
such as fillers and sweetening, flavoring, or coloring agents, may also
be included in these formulations.

[0211] A pharmaceutical composition of the invention may also be prepared,
packaged, or sold in the form of oil-in-water emulsion or a water-in-oil
emulsion. The oily phase may be a vegetable oil such as olive or arachis
oil, a mineral oil such as liquid paraffin, or a combination of these.
Such compositions may further comprise one or more emulsifying agents
such as naturally occurring gums such as gum acacia or gum tragacanth,
naturally-occurring phosphatides such as soybean or lecithin phosphatide,
esters or partial esters derived from combinations of fatty acids and
hexitol anhydrides such as sorbitan monooleate, and condensation products
of such partial esters with ethylene oxide such as polyoxyethylene
sorbitan monooleate. These emulsions may also contain additional
ingredients including, for example, sweetening or flavoring agents.

[0212] Methods for impregnating or coating a material with a chemical
composition are known in the art, and include, but are not limited to
methods of depositing or binding a chemical composition onto a surface,
methods of incorporating a chemical composition into the structure of a
material during the synthesis of the material (i.e., such as with a
physiologically degradable material), and methods of absorbing an aqueous
or oily solution or suspension into an absorbent material, with or
without subsequent drying.

[0213] Controlled- or sustained-release formulations of a composition of
the invention may be made using conventional technology, in addition to
the disclosure set forth elsewhere herein. In some cases, the dosage
forms to be used can be provided as slow or controlled-release of one or
more active ingredients therein using, for example, hydropropylmethyl
cellulose, other polymer matrices, gels, permeable membranes, osmotic
systems, multilayer coatings, microparticles, liposomes, or microspheres
or a combination thereof to provide the desired release profile in
varying proportions. Suitable controlled-release formulations known to
those of ordinary skill in the art, including those described herein, can
be readily selected for use with the compositions of the invention.

[0214] Controlled-release of an active ingredient can be stimulated by
various inducers, for example pH, temperature, enzymes, water, or other
physiological conditions or compounds. The term "controlled-release
component" in the context of the present invention is defined herein as a
compound or compounds, including, but not limited to, polymers, polymer
matrices, gels, permeable membranes, liposomes, nanoparticles, or
microspheres or a combination thereof that facilitates the
controlled-release of the active ingredient.

Administration/Dosing

[0215] The regimen of administration may affect what constitutes an
effective amount. The therapeutic formulations may be administered to the
subject either prior to or after a diagnosis of disease. Further, several
divided dosages, as well as staggered dosages may be administered daily
or sequentially, or the dose may be continuously infused, or may be a
bolus injection. Further, the dosages of the therapeutic formulations may
be proportionally increased or decreased as indicated by the exigencies
of the therapeutic or prophylactic situation.

[0216] Administration of the compositions of the present invention to a
subject, preferably a mammal, more preferably a human, may be carried out
using known procedures, at dosages and for periods of time effective to
prevent or treat disease. An effective amount of the therapeutic compound
necessary to achieve a therapeutic effect may vary according to factors
such as the activity of the particular compound employed; the time of
administration; the rate of excretion of the compound; the duration of
the treatment; other drugs, compounds or materials used in combination
with the compound; the state of the disease or disorder, age, sex,
weight, condition, general health and prior medical history of the
subject being treated, and like factors well-known in the medical arts.
Dosage regimens may be adjusted to provide the optimum therapeutic
response. For example, several divided doses may be administered daily or
the dose may be proportionally reduced as indicated by the exigencies of
the therapeutic situation. A non-limiting example of an effective dose
range for a therapeutic compound of the invention is from about 1 and
5,000 mg/kg of body weight/per day. One of ordinary skill in the art
would be able to study the relevant factors and make the determination
regarding the effective amount of the therapeutic compound without undue
experimentation.

[0217] The compound may be administered to an animal as frequently as
several times daily, or it may be administered less frequently, such as
once a day, once a week, once every two weeks, once a month, or even less
frequently, such as once every several months or even once a year or
less. The frequency of the dose will be readily apparent to the skilled
artisan and will depend upon any number of factors, such as, but not
limited to, the type and severity of the disease being treated, the type
and age of the animal, etc. The formulations of the pharmaceutical
compositions described herein may be prepared by any method known or
hereafter developed in the art of pharmacology. In general, such
preparatory methods include the step of bringing the active ingredient
into association with a carrier or one or more other accessory
ingredients, and then, if necessary or desirable, shaping or packaging
the product into a desired single- or multi-dose unit.

[0218] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of this invention may be varied so as to
obtain an amount of the active ingredient that is effective to achieve
the desired therapeutic response for a particular subject, composition,
and mode of administration, without being toxic to the subject.

[0219] A medical doctor, e.g., physician or veterinarian, having ordinary
skill in the art may readily determine and prescribe the effective amount
of the pharmaceutical composition required. For example, the physician or
veterinarian could start doses of the compounds of the invention employed
in the pharmaceutical composition at levels lower than that required in
order to achieve the desired therapeutic effect and gradually increase
the dosage until the desired effect is achieved.

[0220] In particular embodiments, it is especially advantageous to
formulate the compound in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the subjects to
be treated; each unit containing a predetermined quantity of therapeutic
compound calculated to produce the desired therapeutic effect in
association with the required pharmaceutical vehicle. The dosage unit
forms of the invention are dictated by and directly dependent on (a) the
unique characteristics of the therapeutic compound and the particular
therapeutic effect to be achieved, and (b) the limitations inherent in
the art of compounding/formulating such a therapeutic compound for the
treatment of a disease in a subject.

[0221] In one embodiment, the compositions of the invention are
administered to the subject in dosages that range from one to five times
per day or more. In another embodiment, the compositions of the invention
are administered to the subject in range of dosages that include, but are
not limited to, once every day, every two, days, every three days to once
a week, and once every two weeks. It will be readily apparent to one
skilled in the art that the frequency of administration of the various
combination compositions of the invention will vary from subject to
subject depending on many factors including, but not limited to, age,
disease or disorder to be treated, gender, overall health, and other
factors. Thus, the invention should not be construed to be limited to any
particular dosage regime and the precise dosage and composition to be
administered to any subject will be determined by the attending physical
taking all other factors about the subject into account.

[0222] Compounds of the invention for administration may be in the range
of from about 0.1 mg to about 1,000 mg, about 0.2 mg to about 950 mg,
about 0.4 mg to about 900 mg, about 1 mg to about 850 mg, about 5 mg to
about 750 mg, about 20 mg to about 700 mg, about 30 mg to about 600 mg,
about 50 mg to about 500 mg, about 75 mg to about 400 mg, about 100 mg to
about 300 mg, about 120 mg to about 250 mg, and any and all whole or
partial increments therebetween.

[0223] In some embodiments, the dose of a compound of the invention is
from about 1 mg and about 2,500 mg. In some embodiments, a dose of a
compound of the invention used in compositions described herein is less
than about 10,000 mg, or less than about 8,000 mg, or less than about
6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or
less than about 2,000 mg, or less than about 1,000 mg, or less than about
500 mg, or less than about 200 mg, or less than about 50 mg. Similarly,
in some embodiments, a dose of a second compound (i.e., a drug used for
treating the same or another disease as that treated by the compositions
of the invention) as described herein is less than about 1,000 mg, or
less than about 800 mg, or less than about 600 mg, or less than about 500
mg, or less than about 400 mg, or less than about 300 mg, or less than
about 200 mg, or less than about 100 mg, or less than about 50 mg, or
less than about 40 mg, or less than about 30 mg, or less than about 25
mg, or less than about 20 mg, or less than about 15 mg, or less than
about 10 mg, or less than about 5 mg, or less than about 2 mg, or less
than about 1 mg, or less than about 0.5 mg, and any and all whole or
partial increments thereof.

[0224] In one embodiment, the present invention is directed to a packaged
pharmaceutical composition comprising a container holding a
therapeutically effective amount of a composition of the invention, alone
or in combination with a second pharmaceutical agent; and instructions
for using the composition to treat, prevent, or reduce one or more
symptoms of a disease in a subject.

[0226] Suitable compositions and dosage forms include, for example,
tablets, capsules, caplets, pills, gel caps, troches, dispersions,
suspensions, solutions, syrups, granules, beads, transdermal patches,
gels, powders, pellets, magmas, lozenges, creams, pastes, plasters,
lotions, discs, suppositories, liquid sprays for nasal or oral
administration, dry powder or aerosolized formulations for inhalation,
compositions and formulations for intravesical administration and the
like. It should be understood that the formulations and compositions that
would be useful in the present invention are not limited to the
particular formulations and compositions that are described herein.

Oral Administration

[0227] For oral application, particularly suitable are tablets, dragees,
liquids, drops, suppositories, or capsules, caplets and gelcaps. Other
formulations suitable for oral administration include, but are not
limited to, a powdered or granular formulation, an aqueous or oily
suspension, an aqueous or oily solution, a paste, a gel, toothpaste, a
mouthwash, a coating, an oral rinse, or an emulsion. The compositions
intended for oral use may be prepared according to any method known in
the art and such compositions may contain one or more agents selected
from the group consisting of inert, non-toxic pharmaceutically excipients
that are suitable for the manufacture of tablets. Such excipients
include, for example an inert diluent such as lactose; granulating and
disintegrating agents such as cornstarch; binding agents such as starch;
and lubricating agents such as magnesium stearate.

[0228] Tablets may be non-coated or they may be coated using known methods
to achieve delayed disintegration in the gastrointestinal tract of a
subject, thereby providing sustained release and absorption of the active
ingredient. By way of example, a material such as glyceryl monostearate
or glyceryl distearate may be used to coat tablets. Further by way of
example, tablets may be coated using methods described in U.S. Pat. Nos.
4,256,108; 4,160,452; and 4,265,874 to form osmotically controlled
release tablets. Tablets may further comprise a sweetening agent, a
flavoring agent, a coloring agent, a preservative, or some combination of
these in order to provide for pharmaceutically elegant and palatable
preparation.

[0229] Hard capsules comprising the active ingredient may be made using a
physiologically degradable composition, such as gelatin. Such hard
capsules comprise the active ingredient, and may further comprise
additional ingredients including, for example, an inert solid diluent
such as calcium carbonate, calcium phosphate, or kaolin.

[0230] Soft gelatin capsules comprising the active ingredient may be made
using a physiologically degradable composition, such as gelatin. Such
soft capsules comprise the active ingredient, which may be mixed with
water or an oil medium such as peanut oil, liquid paraffin, or olive oil.

[0231] For oral administration, the compositions of the invention may be
in the form of tablets or capsules prepared by conventional means with
pharmaceutically acceptable excipients such as binding agents; fillers;
lubricants; disintegrates; or wetting agents. If desired, the tablets may
be coated using suitable methods and coating materials such as OPADRY.TM.
film coating systems available from Colorcon, West Point, Pa. (e.g.,
OPADRY.TM. OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric
OY-A Type, OY-PM Type and OPADRY.TM. White, 32K18400).

[0232] Liquid preparation for oral administration may be in the form of
solutions, syrups or suspensions. The liquid preparations may be prepared
by conventional means with pharmaceutically acceptable additives such as
suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated
edible fats); emulsifying agent (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., almond oil, oily esters or ethyl alcohol); and
preservatives (e.g., methyl or propyl para-hydroxy benzoates or sorbic
acid). Liquid formulations of a pharmaceutical composition of the
invention which are suitable for oral administration may be prepared,
packaged, and sold either in liquid form or in the form of a dry product
intended for reconstitution with water or another suitable vehicle prior
to use.

[0233] A tablet comprising the active ingredient may, for example, be made
by compressing or molding the active ingredient, optionally with one or
more additional ingredients. Compressed tablets may be prepared by
compressing, in a suitable device, the active ingredient in a
free-flowing form such as a powder or granular preparation, optionally
mixed with one or more of a binder, a lubricant, an excipient, a surface
active agent, and a dispersing agent. Molded tablets may be made by
molding, in a suitable device, a mixture of the active ingredient, a
pharmaceutically acceptable carrier, and at least sufficient liquid to
moisten the mixture. Pharmaceutically acceptable excipients used in the
manufacture of tablets include, but are not limited to, inert diluents,
granulating and disintegrating agents, binding agents, and lubricating
agents. Known dispersing agents include, but are not limited to, potato
starch and sodium starch glycollate. Known surface-active agents include,
but are not limited to, sodium lauryl sulphate. Known diluents include,
but are not limited to, calcium carbonate, sodium carbonate, lactose,
microcrystalline cellulose, calcium phosphate, calcium hydrogen
phosphate, and sodium phosphate. Known granulating and disintegrating
agents include, but are not limited to, corn starch and alginic acid.
Known binding agents include, but are not limited to, gelatin, acacia,
pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropyl
methylcellulose. Known lubricating agents include, but are not limited
to, magnesium stearate, stearic acid, silica, and talc.

[0234] Granulating techniques are well known in the pharmaceutical art for
modifying starting powders or other particulate materials of an active
ingredient. The powders are typically mixed with a binder material into
larger permanent free-flowing agglomerates or granules referred to as a
"granulation." For example, solvent-using "wet" granulation processes are
generally characterized in that the powders are combined with a binder
material and moistened with water or an organic solvent under conditions
resulting in the formation of a wet granulated mass from which the
solvent must then be evaporated.

[0235] Melt granulation generally consists in the use of materials that
are solid or semi-solid at room temperature (i.e. having a relatively low
softening or melting point range) to promote granulation of powdered or
other materials, essentially in the absence of added water or other
liquid solvents. The low melting solids, when heated to a temperature in
the melting point range, liquefy to act as a binder or granulating
medium. The liquefied solid spreads itself over the surface of powdered
materials with which it is contacted, and on cooling, forms a solid
granulated mass in which the initial materials are bound together. The
resulting melt granulation may then be provided to a tablet press or be
encapsulated for preparing the oral dosage form. Melt granulation
improves the dissolution rate and bioavailability of an active (i.e.
drug) by forming a solid dispersion or solid solution.

[0236] U.S. Pat. No. 5,169,645 discloses directly compressible
wax-containing granules having improved flow properties. The granules are
obtained when waxes are admixed in the melt with certain flow improving
additives, followed by cooling and granulation of the admixture. In
certain embodiments, only the wax itself melts in the melt combination of
the wax(es) and additives(s), and in other cases both the wax(es) and the
additives(s) will melt.

[0237] The present invention also includes a multi-layer tablet comprising
a layer providing for the delayed release of one or more compounds of the
invention, and a further layer providing for the immediate release of a
medication for treatment of a disease. Using a wax/pH-sensitive polymer
mix, a gastric insoluble composition may be obtained in which the active
ingredient is entrapped, ensuring its delayed release.

Parenteral Administration

[0238] As used herein, "parenteral administration" of a pharmaceutical
composition includes any route of administration characterized by
physical breaching of a tissue of a subject and administration of the
pharmaceutical composition through the breach in the tissue.

[0239] Parenteral administration thus includes, but is not limited to,
administration of a pharmaceutical composition by injection of the
composition, by application of the composition through a surgical
incision, by application of the composition through a tissue-penetrating
non-surgical wound, and the like. In particular, parenteral
administration is contemplated to include, but is not limited to,
intraocular, intravitreal, subcutaneous, intraperitoneal, intramuscular,
intrasternal injection, intratumoral, and kidney dialytic infusion
techniques.

[0240] Formulations of a pharmaceutical composition suitable for
parenteral administration comprise the active ingredient combined with a
pharmaceutically acceptable carrier, such as sterile water or sterile
isotonic saline. Such formulations may be prepared, packaged, or sold in
a form suitable for bolus administration or for continuous
administration. Injectable formulations may be prepared, packaged, or
sold in unit dosage form, such as in ampules or in multi-dose containers
containing a preservative. Formulations for parenteral administration
include, but are not limited to, suspensions, solutions, emulsions in
oily or aqueous vehicles, pastes, and implantable sustained-release or
biodegradable formulations. Such formulations may further comprise one or
more additional ingredients including, but not limited to, suspending,
stabilizing, or dispersing agents. In one embodiment of a formulation for
parenteral administration, the active ingredient is provided in dry (i.e.
powder or granular) form for reconstitution with a suitable vehicle (e.g.
sterile pyrogen-free water) prior to parenteral administration of the
reconstituted composition.

[0241] The pharmaceutical compositions may be prepared, packaged, or sold
in the form of a sterile injectable aqueous or oily suspension or
solution. This suspension or solution may be formulated according to the
known art, and may comprise, in addition to the active ingredient,
additional ingredients such as the dispersing agents, wetting agents, or
suspending agents described herein. Such sterile injectable formulations
may be prepared using a non-toxic parenterally-acceptable diluent or
solvent, such as water or 1,3-butanediol, for example. Other acceptable
diluents and solvents include, but are not limited to, Ringer's solution,
isotonic sodium chloride solution, and fixed oils such as synthetic mono-
or di-glycerides. Other parentally-administrable formulations that are
useful include those which comprise the active ingredient in
microcrystalline form, in a liposomal preparation, or as a component of a
biodegradable polymer systems. Compositions for sustained release or
implantation may comprise pharmaceutically acceptable polymeric or
hydrophobic materials such as an emulsion, an ion exchange resin, a
sparingly soluble polymer, or a sparingly soluble salt.

Topical Administration

[0242] A pharmaceutical composition of the invention may be prepared,
packaged, or sold in a formulation suitable for topical administration.
There are several advantages to delivering compounds, including drugs or
other therapeutic agents, into the skin (dermal drug delivery) or into
the body through the skin (transdermal drug delivery). Transdermal
compound delivery offers an attractive alternative to injections and oral
medications. Dermal compound delivery offers an efficient way to deliver
a compound to the skin of a mammal, and preferably a human, and provides
a method of treatment of the skin, or otherwise provides a method of
affecting the skin, without the need to break or damage the outer layer
of the skin. In the present invention, dermal delivery, by way of a
dermally-acting compound of the invention, provides these advantages for
treatment of a skin-related condition, disorder or disease.

[0243] A number of compounds, including some drugs, will penetrate the
skin effectively simply because the molecules are relatively small and
potent at small doses of 0.1 mg to 15 mg/day (Kanikkannan et al., 2000,
Curr. Med. Chem. 7:593-608). Many other compounds and drugs can be
delivered only when an additional enhancement system is provided to
"force" them to pass through the skin. Among several methods of
transdermal drug delivery are electroporation, sonophoresis,
iontophoresis, permeation enhancers (cyclodextrins), and liposomes. While
the aforementioned methods are also included in the present invention for
dermal delivery of the compounds of the invention, liposomes represent a
preferred dermal delivery method.

[0244] The composition of the invention may consist of the active
ingredient alone, in a form suitable for administration to a subject, or
the composition may comprise at least one active ingredient and one or
more pharmaceutically acceptable carriers, one or more additional
ingredients, or some combination of these. The active ingredient may be
present in the composition in the form of a physiologically acceptable
ester or salt, such as in combination with a physiologically acceptable
cation or anion, as is well known in the art. Compositions of the
invention will also be understood to encompass pharmaceutical
compositions useful for treatment of other conditions, disorders and
diseases associated with the skin.

[0245] In one aspect, a dermal delivery vehicle of the invention is a
composition comprising at least one first compound that can facilitate
dermal delivery of at least one second compound associated with, or in
close physical proximity to, the composition comprising the first
compound. As will be understood by the skilled artisan, when armed with
the disclosure set forth herein, such delivery vehicles include, but
should not be limited to, liposomes, nanosomes, phospholipid-based
non-liposome compositions (eg., selected cochleates), among others.

[0246] Formulations suitable for topical administration include, but are
not limited to, liquid or semi-liquid preparations such as liniments,
lotions, oil-in-water or water-in-oil emulsions such as creams, ointments
or pastes, and solutions or suspensions. Topically-administrable
formulations may, for example, comprise from about 0.001% to about 90%
(w/w) active ingredient, although the concentration of the active
ingredient may be as high as the solubility limit of the active
ingredient in the solvent. Formulations for topical administration may
further comprise one or more of the additional ingredients described
herein.

[0247] In one aspect of the invention, a dermal delivery system includes a
liposome delivery system, and that the present invention should not be
construed to be limited to any particular liposome delivery system. Based
on the disclosure set forth herein, the skilled artisan will understand
how to identify a liposome delivery system as being useful in the present
invention.

[0248] The present invention also encompasses the improvement of dermal
and transdermal drug delivery through the use of penetration enhancers
(also called sorption promoters or accelerants), which penetrate into
skin to reversibly decrease the barrier resistance. Many compounds are
known in the art for penetration enhancing activity, including
sulphoxides (such as dimethylsulphoxide, DMSO), azones (e.g.
laurocapram), pyrrolidones (for example 2-pyrrolidone, 2P), alcohols and
alkanols (ethanol, or decanol), glycols (for example propylene glycol,
PG, a common excipient in topically applied dosage forms), surfactants
(also common in dosage forms) and terpenes. Other enhancers include oleic
acid, oleyl alcohol, ethoxydiglycol, laurocapram, alkanecarboxylic acids,
dimethylsulfoxide, polar lipids, or N-methyl-2-pyrrolidone.

[0249] In alternative embodiments, the topically active pharmaceutical or
cosmetic composition may be optionally combined with other ingredients
such as moisturizers, cosmetic adjuvants, anti-oxidants, chelating
agents, surfactants, foaming agents, conditioners, humectants, wetting
agents, emulsifying agents, fragrances, viscosifiers, buffering agents,
preservatives, sunscreens and the like. In another embodiment, a
permeation or penetration enhancer is included in the composition and is
effective in improving the percutaneous penetration of the active
ingredient into and through the stratum corneum with respect to a
composition lacking the permeation enhancer. Various permeation
enhancers, including oleic acid, oleyl alcohol, ethoxydiglycol,
laurocapram, alkanecarboxylic acids, dimethylsulfoxide, polar lipids, or
N-methyl-2-pyrrolidone, are known to those of skill in the art.

[0250] In another aspect, the composition may further comprise a
hydrotropic agent, which functions to increase disorder in the structure
of the stratum corneum, and thus allows increased transport across the
stratum corneum. Various hydrotropic agents such as isopropyl alcohol,
propylene glycol, or sodium xylene sulfonate, are known to those of skill
in the art. The compositions of this invention may also contain active
amounts of retinoids (i.e., compounds that bind to any members of the
family of retinoid receptors), including, for example, tretinoin,
retinol, esters of tretinoin and/or retinol and the like.

[0251] The composition of the invention may comprise a preservative from
about 0.005% to 2.0% by total weight of the composition. The preservative
is used to prevent spoilage in the case of an aqueous gel because of
repeated patient use when it is exposed to contaminants in the
environment from, for example, exposure to air or the patient's skin,
including contact with the fingers used for applying a composition of the
invention such as a therapeutic gel or cream. Examples of preservatives
useful in accordance with the invention included but are not limited to
those selected from the group consisting of benzyl alcohol, sorbic acid,
parabens, imidurea and combinations thereof. A particularly preferred
preservative is a combination of about 0.5% to 2.0% benzyl alcohol and
0.05% to 0.5% sorbic acid.

[0252] The composition preferably includes an antioxidant and a chelating
agent which inhibit the degradation of the compound for use in the
invention in the aqueous gel formulation. Preferred antioxidants for some
compounds are BHT, BHA, alpha-tocopherol and ascorbic acid in the
preferred range of about 0.01% to 5% and BHT in the range of 0.01% to 1%
by weight by total weight of the composition. Preferably, the chelating
agent is present in an amount of from 0.01% to 0.5% by weight by total
weight of the composition. Particularly preferred chelating agents
include edetate salts (e.g. disodium edetate) and citric acid in the
weight range of about 0.01% to 0.20% and more preferably in the range of
0.02% to 0.10% by weight by total weight of the composition. The
chelating agent is useful for chelating metal ions in the composition
which may be detrimental to the shelf life of the formulation. While BHT
and disodium edetate are the particularly preferred antioxidant and
chelating agent respectively for some compounds, other suitable and
equivalent antioxidants and chelating agents may be substituted therefore
as would be known to those skilled in the art.

[0254] A pharmaceutical composition of the invention may be prepared,
packaged, or sold in a formulation suitable for buccal administration.
Such formulations may, for example, be in the form of tablets or lozenges
made using conventional methods, and may, for example, 0.1 to 20% (w/w)
active ingredient, the balance comprising an orally dissolvable or
degradable composition and, optionally, one or more of the additional
ingredients described herein. Alternately, formulations suitable for
buccal administration may comprise a powder or an aerosolized or atomized
solution or suspension comprising the active ingredient. Such powdered,
aerosolized, or aerosolized formulations, when dispersed, preferably have
an average particle or droplet size in the range from about 0.1 to about
200 nanometers, and may further comprise one or more of the additional
ingredients described herein.

Rectal Administration

[0255] A pharmaceutical composition of the invention may be prepared,
packaged, or sold in a formulation suitable for rectal administration.
Such a composition may be in the form of, for example, a suppository, a
retention enema preparation, and a solution for rectal or colonic
irrigation.

[0256] Suppository formulations may be made by combining the active
ingredient with a non-irritating pharmaceutically acceptable excipient
which is solid at ordinary room temperature (i.e., about 20.degree. C.)
and which is liquid at the rectal temperature of the subject (i.e., about
37.degree. C. in a healthy human). Suitable pharmaceutically acceptable
excipients include, but are not limited to, cocoa butter, polyethylene
glycols, and various glycerides. Suppository formulations may further
comprise various additional ingredients including, but not limited to,
antioxidants, and preservatives.

[0257] Retention enema preparations or solutions for rectal or colonic
irrigation may be made by combining the active ingredient with a
pharmaceutically acceptable liquid carrier. As is well known in the art,
enema preparations may be administered using, and may be packaged within,
a delivery device adapted to the rectal anatomy of the subject. Enema
preparations may further comprise various additional ingredients
including, but not limited to, antioxidants, and preservatives.

[0259] Controlled- or sustained-release formulations of a pharmaceutical
composition of the invention may be made using conventional technology,
using for example proteins equipped with pH sensitive domains or
protease-cleavable fragments. In some cases, the dosage forms to be used
can be provided as slow or controlled-release of one or more active
ingredients therein using, for example, hydropropylmethyl cellulose,
other polymer matrices, gels, permeable membranes, osmotic systems,
multilayer coatings, micro-particles, liposomes, or microspheres or a
combination thereof to provide the desired release profile in varying
proportions. Suitable controlled-release formulations known to those of
ordinary skill in the art, including those described herein, can be
readily selected for use with the pharmaceutical compositions of the
invention. Thus, single unit dosage forms suitable for oral
administration, such as tablets, capsules, gel-caps, and caplets, which
are adapted for controlled-release are encompassed by the present
invention.

[0260] Most controlled-release pharmaceutical products have a common goal
of improving drug therapy over that achieved by their non-controlled
counterparts. Ideally, the use of an optimally designed
controlled-release preparation in medical treatment is characterized by a
minimum of drug substance being employed to cure or control the condition
in a minimum amount of time. Advantages of controlled-release
formulations include extended activity of the drug, reduced dosage
frequency, and increased subject compliance. In addition,
controlled-release formulations can be used to affect the time of onset
of action or other characteristics, such as blood level of the drug, and
thus can affect the occurrence of side effects.

[0261] Most controlled-release formulations are designed to initially
release an amount of drug that promptly produces the desired therapeutic
effect, and gradually and continually release of other amounts of drug to
maintain this level of therapeutic effect over an extended period of
time. In order to maintain this constant level of drug in the body, the
drug must be released from the dosage form at a rate that will replace
the amount of drug being metabolized and excreted from the body.

[0262] Controlled-release of an active ingredient can be stimulated by
various inducers, for example pH, temperature, enzymes, water or other
physiological conditions or compounds. The term "controlled-release
component" in the context of the present invention is defined herein as a
compound or compounds, including, but not limited to, polymers, polymer
matrices, gels, permeable membranes, liposomes, or microspheres or a
combination thereof that facilitates the controlled-release of the active
ingredient.

[0263] In certain embodiments, the formulations of the present invention
may be, but are not limited to, short-term, rapid-offset, as well as
controlled, for example, sustained release, delayed release and pulsatile
release formulations.

[0264] The term sustained release is used in its conventional sense to
refer to a drug formulation that provides for gradual release of a drug
over an extended period of time, and that may, although not necessarily,
result in substantially constant blood levels of a drug over an extended
time period. The period of time may be as long as a month or more and
should be a release that is longer that the same amount of agent
administered in bolus form.

[0265] For sustained release, the compounds may be formulated with a
suitable polymer or hydrophobic material that provides sustained release
properties to the compounds. As such, the compounds for use the method of
the invention may be administered in the form of microparticles, for
example, by injection or in the form of wafers or discs by implantation.

[0266] In a preferred embodiment of the invention, the compounds of the
invention are administered to a subject, alone or in combination with
another pharmaceutical agent, using a sustained release formulation.

[0267] The term delayed release is used herein in its conventional sense
to refer to a drug formulation that provides for an initial release of
the drug after some delay following drug administration and that mat,
although not necessarily, includes a delay of from about 10 minutes up to
about 12 hours.

[0268] The term pulsatile release is used herein in its conventional sense
to refer to a drug formulation that provides release of the drug in such
a way as to produce pulsed plasma profiles of the drug after drug
administration.

[0269] The term immediate release is used in its conventional sense to
refer to a drug formulation that provides for release of the drug
immediately after drug administration.

[0270] As used herein, short-term refers to any period of time up to and
including about 8 hours, about 7 hours, about 6 hours, about 5 hours,
about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40
minutes, about 20 minutes, or about 10 minutes and any or all whole or
partial increments thereof after drug administration after drug
administration.

[0271] As used herein, rapid-offset refers to any period of time up to and
including about 8 hours, about 7 hours, about 6 hours, about 5 hours,
about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40
minutes, about 20 minutes, or about 10 minutes, and any and all whole or
partial increments thereof after drug administration.

Kits of the Invention

[0272] The invention also includes a kit comprising a compound useful
within the methods of the invention and an instructional material that
describes, for instance, administering the compound to a subject as a
prophylactic or therapeutic treatment for cancer as described elsewhere
herein. In an embodiment, the kit further comprises a (preferably
sterile) pharmaceutically acceptable carrier suitable for dissolving or
suspending the therapeutic composition, comprising the compound useful
within the methods of the invention, for instance, prior to administering
the molecule to a subject. Optionally, the kit comprises an applicator
for administering the compound.

[0273] Those skilled in the art will recognize, or be able to ascertain
using no more than routine experimentation, numerous equivalents to the
specific procedures, embodiments, claims, and examples described herein.
Such equivalents were considered to be within the scope of this invention
and covered by the claims appended hereto. For example, it should be
understood, that modifications in reaction conditions, including but not
limited to reaction times, reaction size/volume, and experimental
reagents, such as solvents, catalysts, pressures, atmospheric conditions,
e.g., nitrogen atmosphere, and reducing/oxidizing agents, with
art-recognized alternatives and using no more than routine
experimentation, are within the scope of the present application.

[0274] It is to be understood that wherever values and ranges are provided
herein, all values and ranges encompassed by these values and ranges, are
meant to be encompassed within the scope of the present invention.
Moreover, all values that fall within these ranges, as well as the upper
or lower limits of a range of values, are also contemplated by the
present application.

[0275] The following examples further illustrate aspects of the present
invention. However, they are in no way a limitation of the teachings or
disclosure of the present invention as set forth herein.

EXAMPLES

[0276] The invention is now described with reference to the following
Examples. These Examples are provided for the purpose of illustration
only, and the invention is not limited to these Examples, but rather
encompasses all variations that are evident as a result of the teachings
provided herein.

[0277] The materials and methods employed in the experiments and the
results of the experiments presented in this Example are now described.

[0283] The NIH Small Molecule Repository (202,556 compounds) was used for
the primary screening for RAD51 inhibitors. All the compounds were
dissolved in DMSO (Sigma-Aldrich, St. Louis, Mo.); concentrations of
stock solutions were 2.5 mM or 5 mM. In the working solutions the DMSO
concentration added with the stock of compounds was 2% (v/v), unless
indicated otherwise. The compounds for SAR analysis were purchased from
Chembridge Co. (San Diego, Calif.).

[0292] Ethidium bromide (0.5 mg ml.sup.-1) was added to pUC19 supercoiled
dsDNA (2 .mu.g ml.sup.-1) in buffer containing 25 mM Tris acetate, pH
7.5, 20 mM NaCl, and 10 .mu.M EDTA followed by 1 min incubation. The
fluorescence of the sample was measured using a FlouroMax-3 fluorimeter
at an excitation wavelength of 260 nm and an emission wavelength of 546
nm. Then, the small molecule inhibitors were added in increasing
concentrations and allowed to equilibrate for 1 min, followed by the
fluorescence measurement.

Calculation of IC.sub.50 Value for RAD51 Inhibitors:

[0293] IC.sub.50 values were calculated using GraphPad Prism V5.0 software
and the sigmoidal dose-response function. The data were obtained from
three independent repeats of experiments.

[0301] To test the sensitivity of MEF cells to DNA-damaging agents in the
absence or presence of Compound B02 (5 .mu.M), the clonogenic survival
assay was used as described (Essers et al., 1997, Cell 89:195-204).
Briefly, MEF cells were trypsinized and the appropriate number of cells
was replated on 10 cm tissue culture dishes. After overnight culture,
cells were incubated for 1 h in media containing Compound B02 (5 .mu.M),
followed by addition of mitomycin-C (MMC) or cis-dichlorodiamine platinum
(II) (cisDDP) in indicated concentrations and additional incubation for 1
h. Then the cells were washed 3 times with PBS buffer and incubated in
media containing Compound B02 (5 .mu.M) for 7-10 days. Cells were fixed
and stained using staining solution (0.05% crystal violet, 50% methanol
in PBS); colonies were counted using an AlphaImager 3400 (Alpha Innotech
Inc.). The percent of survival was determined as described (Hall &
Giccia, 2005, "Radiobiology for the Radiologist", 6.sup.th Ed.,
Philadelphia, Pa.: Lippincott Williams & Wilkins, pp. 30-45). The percent
of survival obtained from untreated cells was normalized to 100%
survival.

[0304] In RAD51 and RecA-promoted reactions, joint molecules were
deproteinized by addition of SDS to 1% and proteinase K to 880 .mu.g
mL.sup.-1 and incubation for 15 min at 37.degree. C. 0.1 vol of loading
buffer (70% glycerol, 0.1% bromophenol blue) was added and joint
molecules were either analyzed by electrophoresis in 1% agarose-TAE (40
mM Tris-acetate, pH 8.0, and 1 mM EDTA) gels and quantified using a Storm
840 Phosphorlmager (GE Healthcare); or joint molecules were passed twice
through S-400 Spin columns (GE Healthcare) equilibrated with 25 mM
Tris-acetate, pH 7.5, at 23.degree. C., and used as substrates in branch
migration reactions.

Measurement of B02 Binding to RAD51 by SPR:

[0305] Experiments were performed using the ProteOn XPR36 SPR array system
with ProteOn Manager Software version 3.0 (Bio-Rad). ProteOn GLH sensor
chips were preconditioned with two short pulses each (10 s) of 50 mM
NaOH, 100 mM HCl, and 0.5% SDS. The system was then equilibrated with
PBS-T buffer (20 mM Na-phosphate, 150 mM NaCl, and 0.005% Tween 20, pH
7.4). Individual ligand flow channels were activated for 5 min at
25.degree. C. with a mixture of 1-ethyl-3-[3-dimethylaminopropyl
carbodiimide hydrochloride) (0.2 M) and sulfo-N-hydroxysuccinimide (0.05
M). Immediately after chip activation, either RAD51 (6.8 .mu.M in 10 mM
sodium acetate, pH 4.5), RecA (2.6 .mu.M in 10 mM sodium acetate, pH
4.5), or HIV-1NL4-3 capsid protein (0.5 .mu.M in 10 mM sodium acetate, pH
5.0) was injected across ligand flow channels for 5 min at a flow rate of
30 .mu.l min-1. Excess active ester groups on the sensor surface were
capped by a 5-min injection of 1 M ethanolamine-HCl (pH 8.5). This
resulted in the coupling of RAD51, RecA, and CA to 14,000, 9,000, and
17,000 RUs (response unit, which is an arbitrary unit that corresponds to
1 pg/mm2), respectively. The standard deviation in the immobilization
level from the six spots within each channel was less than 4%. B02 in
indicated concentrations in buffer S containing 25 mM Tris-acetate, pH
7.5, 100 .mu.M CaCl.sub.2, 3% DMSO, and 100 .mu.M ATP (when indicated)
was injected over the control or RAD51 chips at a flow rate of 100 .mu.l
min-1, for a 2-min association phase, followed by a 15-min dissociation
phase at 25.degree. C. using the "one-shot" functionality of the ProteOn.
Specific regeneration of the surfaces between injections was not needed
owing to the nature of the interaction. Data were analyzed using the
ProteOn Manager Software version 3.0 (Bio-Rad). The responses of a buffer
injection and responses from the reference flow cell were subtracted to
account for nonspecific binding. Experimental data were fitted globally
to a simple 1:1 binding model. The average kinetic parameters
(association [ka] and dissociation [kd] rates) generated from three data
sets were used to define the equilibrium dissociation constant (K.sub.d).

[0307] To suppress RAD51 expression, HEK cells were transfected with 100
nM RAD51 siRNA (sc-36361; Santa Cruz Biotechnology). Briefly, cells
(1.times.10.sup.6) were seeded in a 3.5-cm tissue culture plate and
incubated overnight, then the medium was removed and the cell layer was
washed three times with PBS. Transfection was carried out by adding 1 ml
transfection solution containing 100 pmol (1.25.mu.m) siRNA and 10 .mu.l
siRNA transfection reagent (sc-29528; Santa Cruz Biotechnology) in
Opti-MEM I Reduced-Serum medium (Invitrogen) to each plate. Six to 7
hours after transfection the medium was replaced with fresh DMEM+.
Twenty-four, 48, 72, and 96 h after transfection, the RAD51 level was
determined using Western blotting. As a specificity control scrambled
siRNA (sc-37007; Santa Cruz Biotechnology) was used instead of RAD51
siRNA.

[0309] A FRET-based DNA strand exchange assay suitable for HTS of large
libraries of chemical compounds was developed. In this assay, RAD51
promotes DNA strand exchange between homologous synthetic ssDNA and dsDNA
substrates. The dsDNA carries fluorescein (FLU), a fluorescent donor
group, and black hole quencher 1 (BHQ1), a non-fluorescent acceptor
group, which were attached to the 5'- and 3'-ends of the complementary
ssDNA strands, respectively (FIG. 1A). In this dsDNA substrate, the
fluorescence of the FLU group is quenched by BHQ1 through FRET. As a
result of RAD51-promoted DNA strand exchange, the FLU-carrying DNA strand
is displaced from the dsDNA that carries the BHQ1 and the fluorescence of
the FLU group increases (Parkhurst & Parkhurst, 1995, Biochem.
34:293-300; Parkhurst et al., 2001, Biopol. 61:180-200).

[0310] Using this assay the kinetics of RAD51-promoted DNA strand exchange
was measured. RAD51 was loaded on the homologous ssDNA (SEQ ID NO:2;
48-mer) (denoted as "Homologous DNA") to form the nucleoprotein filament.
Then, fluorescently labeled dsDNA (SEQ ID NO:4-Black Hole Quencher 1; and
fluorescein-SEQ ID NO:3) was added to the filament to initiate DNA strand
exchange. After a 1 h incubation the fluorescence intensity at 521 nm
increased approximately 20-fold (FIG. 1B). To ensure that the observed
fluorescence increase resulted from DNA strand exchange, a control was
run in which the RAD51 filament was assembled on heterologous ssDNA (SEQ
ID NO:5, 48-mer) (denoted as "Heterologous DNA"). Since DNA strand
exchange does not occur between heterologous DNA molecules (Shibata et
al., 1979, Proc. Natl. Acad. Sci. 76:1638-42), no increase in
fluorescence was expected. Indeed, in the case of heterologous DNA the
intensity of fluorescence remained almost constant during the 1 h of
incubation (FIG. 1B). Thus, the results validated the FRET-based assay to
measure the DNA strand exchange activity of RAD51.

Example 2

HTS of the NIH Small Molecule Repository

[0311] The NIH Small Molecule Repository (202,556 compounds) was screened
for RAD51 inhibitors using the FRET-based assay described above. 174
positive hits that showed more than 30% inhibition of the DNA strand
exchange activity of RAD51 were detected (hit rate, 0.09%) using a Perkin
Elmer Envision 2102 multilabel reader. Measuring the concentration
dependence of RAD51 inhibition by these compounds allowed the
identification of the seventeen most potent inhibitors as candidates that
warranted further analysis (FIGS. 2A-2B).

Example 3

Analysis of RAD51 Inhibitors Using the D-loop Assay

[0312] To validate the hits identified in the primary FRET-based assay,
the seventeen selected compounds (FIGS. 2A-2B) were further analyzed
using the D-loop assay. In the D-loop assay, DNA strand exchange was
promoted by RAD51 between homologous .sup.32P-labeled ssDNA and pUC19
supercoiled plasmid DNA (FIG. 3A). The products of this reaction, joint
molecules, also known as D-loops (called after the displaced DNA strand
that is formed in joint molecules during DNA strand exchange), were
identified by electrophoresis in a 1% agarose gel. First, the inhibitory
effect of each of the seventeen compounds on the efficiency of D-loop
formation was measured. Eleven of the seventeen compounds inhibited
D-loop formation by more than 50% (FIGS. 3B and 3C, Table 1). Thus, 65%
of the compounds identified in the primary assay were validated by the
secondary assay. In addition, using a fluorescence intercalator (ethidium
bromide) displacement assay, Compounds A05, A06, A14, and A15 were found
to be DNA binders (data not shown). These compounds along with Compounds
A08, A11, and B01, which showed relatively modest inhibition, were not
analyzed further. Then, the IC.sub.50 values for the four most potent
remaining inhibitors of the RAD51 DNA strand exchange activity were
determined using the D-loop assay. The IC.sub.50 for Compounds A03, A04,
A10, and B02 were 33.2 .mu.M, 5.0 .mu.M, 26.6 .mu.M, and 27.4 .mu.M,
respectively (FIG. 4; Table 2).

Analysis of a RAD51 Inhibitor Using the Homologous Pairing and Three
Strand Exchange Assay

[0313] The effect of Compound B02 on homologous pairing activity of RAD51
by D-loop assay was analyzed. RAD51 can promote homologous searching and
pairing of ssDNA on supercoiled plasmid dsDNA, which contains homologous
sequences of ssDNA (FIG. 3A). In this assay, .sup.32P-labeled ssDNA (SEQ
ID NO:6, 90 mer) (0.9 .mu.M, nt) and supercoiled dsDNA (pUC19, 15 .mu.M)
was employed as DNA substrates. Compound B02 was found to efficiently
inhibit the strand exchange promoted by RAD51 with a
concentration-dependent manner (FIG. 3D, lane 1-9). And also
interestingly, the Compound B02 did not inhibit D-loop formation of RecA
protein, the prokaryotic homolog of RAD51 (FIG. 3D, lane 10-18).
IC.sub.50 for RAD51 is 10.4 .mu.M; while for RecA protein is more than
100 .mu.M (FIG. 3E), which indicates that Compound B02 is a very specific
inhibitor for RAD51.

[0314] A strong inhibition of Compound B02 on RAD51 was also observed in
three strand exchange assay, in which a strand exchange between circular
.phi.X174 ssDNA and homologous linearized .phi.X174 dsDNA (linearized by
ApaL1 endonuclease) was promoted by RAD51 (FIG. 10A). Yields of joint
molecules (JM) and nicked circular DNA (NC) were compared among the
different doses of treatment with Compound B02 (FIG. 10B). The results
show that formation of both joint molecules and nicked circular DNA
decreased with an increasing of Compound B02 concentration. IC.sub.50 for
the whole product (JM+NC) was 26.03 .mu.M, which was comparable with the
value observed in D-Loop assay if 1 .mu.M RAD51 was used (data was not
shown). IC.sub.50 for the JM was 23.3 .mu.M and for NC was 26.7 .mu.M,
suggesting that the Compound B02 has the same inhibition to JM and NC
formation.

Example 5

Specificity of RAD51 Inhibitors

[0315] RAD51 shares structural and functional similarity with RecA from E.
coli; both proteins promote DNA strand exchange in vitro and share 30%
homology. Using the D-loop assay, the effect of the selected RAD51
inhibitors on the DNA strand exchange activity of RecA was evaluated.
Compound A03 showed some moderate specificity for RAD51 (FIG. 5A), with
the IC.sub.50 5.6-fold lower for RAD51 than for RecA (Table 2). Compounds
A04 and A10 inhibited RAD51 and RecA with a nearly equal efficiency
(FIGS. 5B & 5C; Table 2). Finally, Compound B02 showed the highest
specificity for RAD51 (FIG. 5D); the IC.sub.50 for RAD51 was 27.4 .mu.M,
whereas for RecA no significant inhibition of DNA strand exchange was
observed up to 250 .mu.M of Compound B02 (Table 2).

[0316] The inhibitory effects of Compounds A03, A04, and A10 compounds
were evaluated to determine whether they are specific for the proteins of
the Rad51/RecA family or have a broader specificity. To address this
question the effect of the inhibitors on human RAD54, a Swi2 protein,
which does not share structural homology with the proteins of the
RecA/RAD51 family, were tested (Mazin et al., 2010, DNA Repair (Amst)
9:286-302). RAD54 promotes branch migration of Holliday junctions, a
process in which one DNA strand is progressively exchanged for another
(FIG. 6A). The effect of Compounds A03, A04, and A10 compounds on the
RAD54 branch migration activity was tested using a .sup.32P-labeled
oligonucleotide-based cruciform DNA substrate, known as the partial
Holliday junction or PX-junction (FIG. 6A). Of the four compounds tested,
Compounds A03, A10 and B02 were shown not to significantly inhibit RAD54
in the range of concentrations from 0 to 200 .mu.M (FIG. 6B). However,
Compound A04 did have an inhibitory effect on the RAD54 branch migration
activity (IC.sub.50=2.6 .mu.M) (FIG. 6C).

[0317] Thus among tested compounds, Compound B02 was identified as a
specific inhibitor of human RAD51. Compounds A03 and A10 inhibited both
RAD51/RecA family proteins, RAD51 and RecA. Compound A04 showed the
broadest inhibitory spectrum by inhibiting all three tested proteins:
RAD51, RecA, and RAD54.

Example 6

Blockage of the RAD51-ssDNA Filament Formation at the Presynaptic Stage

[0318] To explore the mechanism how Compound B02 inhibits the homologous
pair and strand exchange, three experiments were carried out.

[0319] Firstly, the time course of D-loop formation was compared with
different order of addition of Compound B02 and ssDNA (FIG. 11A). In
Protocol (I), 20 .mu.M Compound B02 was added after the filament
formation; and then the samples were incubated for indicated times before
the initiation of D-loop formation by addition of dsDNA. In Protocol
(II), RAD51 was first incubated with 20 .mu.M Compound B02 for indicated
time; and then ssDNA was added and incubated for 15 min at 37.degree. C.
to form the filament before the initiation of D-loop formation by
addition of dsDNA. The results show that, in both Protocols (I) and (II),
the efficiency of D-loop formation decreased in the presence of Compound
B02 (FIG. 11B, lane 1-7 and 8-14), while the efficiency of D-loop
formation increased or keep stable in the absence of Compound B02 (data
not shown). Furthermore, the relative inhibition (FIG. 11C), which was
expressed as the ratio of the joint molecules with Compound B02 treatment
to those without Compound B02 treatment, shown that in the presence of
Compound B02 the efficiency of D-loop formation in Protocol II (FIG. 11B,
lane 8-14) reduced much faster than that in Protocol I (FIG. 11B, lane
1-7).

[0320] The results suggest that Compound B02 can either block the assembly
of the filaments if added before filament formation or disrupt the
assembled filaments if added after the filament formation. However, a
lower portion of filaments was disrupted if Compound B02 was added after
the filament formation, owing to the high stability of nucleoprotein
filaments.

[0321] Secondly, the effect of Compound B02 on assembly of RAD51-ssDNA
filaments was tested using ssDNA binding assay. In this assay, RAD51 (1
.mu.M) was incubated with 25 .mu.M Compound B02, which was comparable
with IC.sub.50 obtained from D-loop assay if 1 .mu.M RAD51 was used (data
was not shown). Then ssDNA (SEQ ID NO:6, 90 mer) (3 .mu.M, nt) was added
in the presence of indicated concentration of NaCl. The binding of ssDNA
on RAD51 protein was impaired with the challenge of increasing
concentration of NaCl. If Compound B02 could disrupt the RAD51-ssDNA
filaments, more .sup.32P-labeled free ssDNA dissociating from the
filaments would be observed. The results (FIGS. 12A-12B) show that
without Compound B02, the ratio of ssDNA in complex decreased from 100%
(0 mM NaCl) to 71% (200 mM NaCl), only 29% loss of filaments; while with
Compound B02, the ratio of ssDNA decreased from 80% (0 mM NaCl) down to
almost 0% (200 mM NaCl), which means all the filaments were disrupted in
the presence of 200 mM NaCl. Besides, even without NaCl challenge, there
was still 20% loss of filaments in the presence of Compound B02. These
results suggest that Compound B02 can disrupt the RAD51-ssDNA filaments.

[0322] Thirdly, the effect of Compound B02 on the ATPase activity of RAD51
was tested by concentration titration of Compound B02. As illustrated in
FIG. 13, Compound B02 can inhibit the ATPase activity of RAD51 with a
concentration-dependent manner, and IC.sub.50 is 37.6 .mu.M. The
DNA-dependent ATPase activity of RAD51 is correlative with the binding of
DNA on the protein. Therefore, the inhibition of Compound B02 on ATPase
activity of RAD51 also suggests that Compound B02 can disrupt the
RAD51-ssDNA filaments.

Example 7

[0323] Disruption of the Binding of dsDNA on RAD51-ssDNA Filament

[0324] In the RAD51 protein, there are two specific DNA-binding sites: the
primary DNA binding site and the secondary binding site (Sung et al.,
2003, J. Biol. Chem. 278:42729-32). In the presynaptic stage of strand
exchange, the RAD51-ssDNA filament was assembled by binding of ssDNA into
primary binding site of RAD51. The search of DNA homology was done by
reiterative binding and release of duplex DNA on and from the secondary
binding site of RAD51 in RAD51-ssDNA filaments until the homology was
located. So the secondary binding site is also indispensable for the
strand exchange. DNA coaggregation assay was used to test the effect of
Compound B02 on binding of duplex DNA on the secondary binding site of
RAD51.

[0325] In this assay, the RAD51-ssDNA filament was assembled by incubation
of 1 .mu.M RAD51 with 3 .mu.M ssDNA (SEQ ID NO:7, 94 mer). RAD51 primary
binding site was saturated by ssDNA at this DNA concentration. Then the
filament was mixed with 25 .mu.M dsDNA (pUC19, linearized by BamHI) and
challenged by NaCl in the absence or presence of 20 .mu.M Compound B02.
If Compound B02 can disrupt the binding of dsDNA on secondary binding
site of RAD51, the DNA coaggreations will be more unstable in Compound
B02 presence samples than in Compound B02 absence samples, and the
percent of dsDNA in coaggregations (FIGS. 14A-14B) will be lower in
Compound B02 presence samples than in Compound B02 absence samples. As
expected, much lower percent of dsDNA in coaggregations was observed in
Compound B02 presence samples. Even without the challenge of NaCl, the
ratio of dsDNA coaggregations in Compound B02 presence samples was only
8%, which is 1/5 of that in Compound B02 absence samples. These results
suggest that Compound B02 can disrupt not only the binding of ssDNA on
primary binding site of RAD51 but also the binding of dsDNA on secondary
site of RAD51, both of which are critical to the homologous
recombination.

Example 8

Inhibition of HDR of a Chromosomal DSB

[0326] DR-GFP assay can be used to monitor HDR of chromosomal DSB by
fusion a DR-GFP report gene construct in chromosome of cells. In DR-GFP
construct (FIG. 15A), SceGFP gene, which encodes green fluorescent
protein (GFP), was disabled by insertion of an 18-bp recognition site for
I-SceI cleavage into SceGFP gene. A DSB can be generated by expressing of
the transfected pCBASce plasmid, which encodes I-SceI endonuclease.
Repair of the I-SceI induced DSB by recruitment of iGFP (an internal GFP
gene fragment truncated at both ends) as a template (FIG. 8A) gives rise
to a functional GFP gene. This HDR event can be scored by green
fluorescence in individual cells, using flow cytometric analysis.
Consequently the effect of Compound B02 on the HDR of a chromosome in
cells can be clearly addressed. The pCBASce plasmid was transfected in
DR-GFP 293 cells in the presence of 0 .mu.M, 5 .mu.M, 10 .mu.M and 20
.mu.M of Compound B02. To validate the gene transfection, the cells
transfected with pMX-GFP, which encodes functional GFP protein, were used
as a positive control. And untransfected parental cells were used as a
negative control. Compound B02 can reduce the yield of GFP positive cells
by a concentration-dependent manner. As shown in FIG. 15B, the yield of
GFP positive cells decreased from 3.28% in non-treatment cells to 0.40%
in the cells treated with 20 .mu.M of Compound B02, an 8-fold reduction
was observed, suggesting that as a RAD51 inhibitor, Compound B02 can
efficiently disrupt the HDR promoted by RAD51. But this claim can be
easily invalidated by two possibilities: (1) Compound B02 inhibits the
transfection or expression of pCBASce, which just produces less DSB than
that in the absence of Compound B02, and consequently less DSB repair
will produce lower level of GFP positive cells; (2) Compound B02 inhibits
the expression of RAD51 in the cells. Recently published paper has
reported a Histone deacetylase (HDAC) inhibitor, which indirectly
inhibited HDR by reducing the expression of RAD51 (Adimoolan et al.,
2007, Proc. Natl. Acad. Sci. USA). To exclude these possibilities, first
the effect of Compound B02 on the plasmid transfection was tested. The
pMX-GFP plasmid, which encodes the functional GFP protein, was used as a
reporter. After transfection of pMX-GFP plasmid into DR-GFP 293 cells,
the expression of functional GFP protein was monitored by flow cytometric
analysis. The cells transfected with pMX-GFP plasmid, but without
treatment of Compound B02 were used as a control. It was observed that
Compound B02 has no inhibition to transfection in comparison to control
cells. The ratio of GFP positive cells is comparable between the cells in
control group (31.52%) and the cells with 20 .mu.M Compound B02 (31.47%)
(FIG. 15C). Furthermore, the expression of I-SceI protein in DR-GFP 293
cells was assessed with or without treatment of Compound B02 using
western blot assay. The results show that Compound B02 did not affect the
expression of I-SceI protein (FIGS. 16A & 16B). Next, a western blot
assay was carried out to test the effect of Compound B02 on the
expression of RAD51, the data show that the expression of RAD51 was
comparable between the cells without treatment of Compound B02 and cells
treated with 20 .mu.M Compound B02 (FIGS. 16C and 16D). The results of
the two experiments above suggest that the reduction of yield of GFP
positive cells is attributed to the inhibition of Compound B02 on RAD51
protein, which is a key protein in the HDR of Chromosomal DSB.

Example 9

Sensitivity of MEF Cells to Double-Strand Break (DSB) Induced Agents

[0327] Based on the observation that Compound B02 can inhibit the
homologous recombination in vitro, it is quite interesting to explore if
Compound B02 has inhibition on homologous recombination in cell level.
Herein the sensitivity of MEF cell lines (wide type and Tp53-/-) to the
DNA cross-linking agents cisDDP and MMC in the absence and presence of
Compound B02 was analyzed by using colongenic survival assay. Before this
experiment, the toxicity of Compound B02 to the cell lines selected was
first assessed. No decrease of cellular viability with the treatment up
to 5 .mu.M Compound B02 was observed. Therefore this concentration was
selected for the colongenic survival assay. As shown in FIGS. 17A-17C, in
the presence of 5 .mu.M Compound B02, both cell lines were more sensitive
to the cisDDP and MMC, which suggest that Compound B02 may reduce the
resistance of cells to DNA damage agents by inhibiting the DSB repair.
Some SSB-induced agents such as MMS induce DNA damage. Failure to repair
the SSB could produce stalled replication fork, and collapsing of the
stalled replication fork could finally cause DSB. PARP-1 protein is a key
protein in BER pathway, which is very important in SSB repair. Here to
validate inhibition of Compound B02 to RAD51 protein in mammalian cells,
the sensitivity of MEF cells to DNA damage agents MMS was tested. MEF
cells were treated by MMS to induce the SSBs in the presence of 5 .mu.M
Compound B02 or 1 .mu.M of a PARP-1 inhibitor, AZD2281 (olaparib or
4-[(3-[(4-cyclopropylcarbonyl)piperazin-4-yl]carbonyl)
-4-fluorophenyl]methyl(2H)phthalazin-1-one) or both. The cells treated
only with MMS were used as a control. The results show that in the
presence of 1 .mu.M AZD2281, the cells are more sensitive to MMS than
that in the absence of AZD2281 (FIG. 18). The sensitivity enhancement may
be attributed to that the treatment of AZD2281 inhibits the BER, which
blocks the repair of the SSB induced by MMS. Additionally, if in the
presence of both Compound B02 and AZD2281, the cells were more sensitive
to MMS than that only in the presence of AZD2281, suggesting that not
only the SSB repair was blocked, meanwhile the repair of DSB induced by
the collapsing the stalled fork was also blocked, the data shows evidence
that the Compound B02 inhibits the repair of DSB in the cells by
inhibition of the RAD51 activity. Also the enhancement of sensitivity was
observed in the cells with co-treatment of the MMS and Compound B02. This
could happen because even in the non-treated cells there is certain
amount of stalled fork which could collapse and induce DSB. The
enhancement of sensitivity may response to the inhibition of Compound B02
to DSB repair.

Example 10

SAR Analysis of Compound B02

[0328] For SAR analysis of Compound B02, a 16-compound library of B02
derivatives was selected (FIG. 7A). According to the structural features,
the 16 compounds were sorted in 5 groups. In group 1(compound C1):
(E)-2-(pyridin-3-yl) vinyl was removed; in group 2 (compounds C2a to
C2h): (E)-2-(pyridin-3-yl) vinyl was replaced with
(E)-2-(R.sub.1-substituting group) vinyl; in group 3 (compounds C3a to
C3e): benzyl was replaced with R.sub.2; in group 4 (compound C4): the
core, quinazolin-4(3H)-one was replaced by 6-iodo-quinazolin-4(3H )-one;
in group 5 (compound C5): the core, quinazolin-4(3H)-one was replaced by
6-iodo-quinazolin-4(3H)-one, as well as (E)-2-(pyridin-3-yl) vinyl was
replaced by (E)-2-(pyridin-2-yl) vinyl.

[0329] The inhibitory effect of these B02 derivatives (50 .mu.M) on the
DNA strand exchange activity of RAD5 was determined using the D-loop
assay. The only position of BO2 that could tolerate modifications and
still inhibit RAD51 was the benzyl (compound group 3) (FIGS. 7B and 7C).
Moreover, only compound C3a (R.sub.2=ethyl) and compound C3b
(R.sub.2=m-methyl phenyl) retain the inhibitory effect, the substitutions
of other groups (C3d, R.sub.2=phenyl and C3e, R.sub.2=methyl) or at the
different positions (C3c, R.sub.2=p-methyl phenyl) eliminates the
inhibition, suggesting that the inhibition is tightly related to the size
and the steric conformation of the substituting group. All other tested
replacements in the groups 1, 2, 4, and 5 eliminated RAD51 inhibition.
The high sensitivity of the RAD51 inhibition to B02 modifications
suggests specific interactions between the inhibitor molecule and RAD51
protein.

Example 11

Inhibitor Optimization

[0330] From the library of B02 derivatives, two compounds (C3a and C3b)
that inhibited RAD51 were identified (FIGS. 7A-7C). For these two
inhibitors, the IC.sub.50 values of the RAD51 DNA strand exchange
activity and their selectivity for RAD51 were determined using the D-loop
assay (FIGS. 8A-8C). The IC.sub.50 for C3a and C3b are 15.3 .mu.M and
27.3 .mu.M, respectively (FIGS. 8B & 8C). To evaluate the selectivity of
the inhibitors the effect of C3a and C3b on the DNA strand exchange
activity of RecA were determined. The results show that compounds C3a and
C3b in concentrations up to 100 .mu.M do not inhibit RecA (FIGS. 8A-8C;
Table 2).

[0331] Thus, the current results demonstrate the feasibility and
efficiency of the HTS approach for discovery of novel selective
inhibitors of RAD51, a key protein of homologous recombination and the
repair of DNA double strand breaks and interstrand crosslinks. Further
experiments may include establishing the mechanism of specific RAD51
inhibition by selected small molecule compounds (B02, A03, A10) and
examining the effect of these compounds on the RAD51-dependent homologous
recombination and DNA repair in human cells.

Example 12

Binding to RAD51 and Inhibition of its Activities

[0332] RAD51 and its E. coli homologue RecA possess DNA strand exchange
and DNA branch migration activities. The effect of B02 (FIG. 19A) on
these activities of both proteins was examined. pBSK (+) gapped and
linear dsDNA substrates that allow separate analysis of DNA strand
exchange and branch migration promoted by RAD51/RecA were used (FIG.
19B). At the first step, RAD51/RecA promoted DNA strand exchange between
gapped DNA and linear DNA substrates resulting in formation of joint
molecules. Joint molecules were then purified and used as substrates for
RAD51/RecA branch migration. In accord with previous data showing
specific inhibition of RAD51 by B02 in the D-loop assay, B02 (10-100
.mu.M) inhibited DNA strand exchange promoted by RAD51 (FIGS. 19C-19D),
but not by RecA (FIGS. 19D & 20A). B02 (10-100 .mu.M) was found to
inhibit the DNA branch migration activity of RAD51 (FIGS. 19E-19F). The
inhibition was specific, as B02 did not inhibit the branch migration
activity of RecA (FIGS. 19F & 20B). The IC.sub.50 value of RAD51
inhibition by B02 was 35 .mu.M for both DNA strand exchange and DNA
branch migration. Next, it was tested whether RAD51 inhibition is caused
by the direct interaction of B02 with RAD51. Using the surface plasmon
resonance (SPR) technique, B02 (6.25-50 .mu.M) was shown to bind to
RAD51, but not to RecA (FIGS. 21 & 22A-22D). For B02 binding to RAD51 in
the absence of ATP, kinetic values were as follows: k.sub.a=4.5
(.+-.0.3).times.10.sup.3 M.sup.-1s.sup.-1; k.sub.d=2.5
(.+-.0.3).times.10.sup.-2 s.sup.-1; K.sub.d=5.6 .mu.M. Using the ethidium
bromide displacement assay B02 was shown not to bind DNA. Thus, B02
inhibited DNA strand exchange and branch migration activities through
direct and specific binding to RAD51.

Example 13

Disruption of the RAD51 Foci Formation

[0333] B02 was tested to determine whether it can inhibit RAD51 activities
in the cell. In response to DNA damage, RAD51 accumulates in distinct
nuclear structures, known as foci. Because RAD51 foci colocalize with
ssDNA formed in the cell after DNA damage, it is thought that the foci
represent RAD51 complexes with DNA repair intermediates. B02 was found to
inhibit RAD51 foci formation induced in 293 human embryonic kidney (HEK)
cells by IR. In the presence of B02 (50 .mu.M), the fraction of cells
with RAD51 foci (.gtoreq.1 focus) was decreased 3.8-fold, from 72.+-.10%
to 19.+-.6%, almost to the level of foci formation observed in
non-irradiated cells (15.+-.13%) (FIGS. 23A-23B); the average number of
RAD51 foci per nucleus decreased 4.4-fold, from 53.+-.11 to 12.+-.4 (FIG.
23C). At lower concentrations (20 .mu.M), B02 also inhibited IR-induced
RAD51 foci formation, however the inhibitory effect was smaller.

Example 14

Increase in Cell Sensitivity to DNA-Damaging Agents

[0334] B02 was examined to determine whether it can enhance cell
sensitivity to DSB- and ICL-inducing anticancer agents, cisplatin and
MMC. Using the clonogenic survival assay it was found that in the
presence of B02 (5 .mu.M) mouse embryonic fibroblasts (MEF) became
approximately 17- and fivefold more sensitive to cisplatin (32 .mu.M) and
MMC (1 .mu.M), respectively (FIGS. 24A-24B). Because, p53 protein is
commonly mutated in many human cancers, the effect of B02 on Tp53.sup.-/-
MEF was also tested. It was found that the sensitivity of Tp53.sup.-/-
MEF to cisplatin and MMC increased in the presence of B02 similarly to
wild type cells (FIGS. 25A-25B). In these experiments, 5 .mu.M B02 was
used, a concentration at which B02 alone did not have a substantial
effect on the viability of wild type or Tp53.sup.-/- MEF (FIG. 24C).
Inhibitory effect of B02 on cell survival observed in co-treatment
experiments could be due to depletion of RAD51 that translocated to the
sites of DNA damage. This hypothesis was tested using HEK cells in which
the RAD51 expression level was decreased by siRNA (FIGS. 26A-26B). The
results showed that indeed the combination of specific RAD51 siRNA and
B02 more strongly sensitized HEK cells to cisplatin than did each of
these reagents alone (FIG. 24D). The minimum incubation time with B02
that was required for cells' sensitization for cisplatin was determined.
These data indicate that 10-12 h of incubation was required to increase
the sensitivity of HEK cells for cisplatin (FIG. 24E). After 16 h, the
maximal sensitivity was reached. Thus, B02 causes cell sensitization to
cisplatin and MMC, indicating the ability of B02 to inhibit
RAD51-dependent DSB repair in the cell.

[0335] As described herein, the mechanism of RAD51 inhibition by B02 and
its ability to inhibit RAD51 homologous recombination and DNA repair in
cells was analyzed. B02 was found to bind to RAD51 and inhibit its DNA
strand exchange and branch migration activities with high specificity, as
it does not affect E. coli RecA, a structural and functional homologue of
RAD51. Importantly, these results demonstrated that B02 can inhibit
RAD51-dependent HR events in the cell and promote cell killing by
cytotoxic DSBand ICL-inducing agents.

[0336] Because the RAD51-ssDNA filament plays a critical role in HR, its
formation is tightly regulated by various factors that either enhance or
inhibit RAD51 binding to ssDNA. These data demonstrated that B02 also
inhibits RAD51 filament formation. These results showed that B02 impairs
RAD51 filament formation by targeting protein-DNA interactions. The
filament formation involves binding ssDNA to the RAD51 primary site.
Using a coaggregation assay, it was found that B02 also inhibits dsDNA
binding to the secondary RAD51 site, which normally occurs during the
search for homology.

[0337] To address of whether B02 inhibits RAD51-dependent HR and DNA
repair in the cell, several cell-based assays were carried out. First, it
was found that B02 inhibited formation of RAD51 foci in response to IR,
which is thought to reflect RAD51 accumulation at the sites of damaged
DNA and formation of RAD51-DNA complexes during recombinational DNA
repair. Then, using a chromosomally integrated GFP reporter, it was shown
that B02 decreased, up to eightfold, the frequency of DSB-induced HR in
human cells. It was also demonstrated that B02 increased cell sensitivity
to ICL- and DSB-inducing agents, cisplatin and MMC. Finally, it was found
that a combination of B02 with PARP 1 inhibitor AZD2281 increased cell
sensitivity to an alkylating agent (MMS) to a greater extent than does
AZD2281 alone. The finding that PARP 1 inhibitors enhance the effect of
B02 on cell sensitivity to DNA damage is consistent with inhibition of HR
by B02. In the co-treatment experiments, B02 showed activity at lower
concentrations (5 .mu.M) than in other biological assays, likely due to
the depletion of RAD51 that accumulates at the sites of DNA damage.
Indeed, it was found that depletion of RAD51 with siRNA had an additive
effect with B02 treatment. Moreover, RAD51 is not a canonical enzyme; the
DNA strand exchange assay requires rather high RAD51 concentrations
(stoichiometric relative to DNA substrates). Concerning RAD51 foci
formation, it is worth noting that each RAD51 focus involves thousands of
RAD51 monomers, therefore a decrease in their number in each focus may
not have been readily detectable by immunostaining and required higher
B02 concentrations (20-50 .mu.M). Overall, these results demonstrated
that B02 inhibits RAD51-dependent HR and DSB repair in mammalian cells
(FIG. 27).

[0338] Because small-molecule inhibitors may be applied in a cell cycle
and in a concentration dependent manner, they are especially useful for
analysis of proteins essential for cell viability, like RAD51. By
applying B02 for different periods of time after DNA damage by cisplatin,
a maximal time for which DNA repair can be delayed before the cells start
dying was determined.

[0339] These results indicated that a combination of inhibitors that
target alternative DNA repair pathways, e.g., RAD51-dependent and
PARP1-dependent DNA repair, can be especially efficient for sensitizing
cancer cells for radio- and chemotherapeutic agents. Targeting RAD51 may
represent an important strategy to specifically eradicate cancer cells.
Consistent with the compensatory role that HR may play in cancer cells,
RAD51 was found to be overexpressed in many tumors.

[0340] These results demonstrate that B02 inhibitor of RAD51 can
efficiently suppress DSB dependent HR in the cell. The inhibitor can be
used for the analysis of RAD51 cellular functions and for development of
novel anticancer therapies.

[0341] The disclosures of each and every patent, patent application, and
publication cited herein are hereby incorporated herein by reference in
their entirety.

[0342] While the invention has been disclosed with reference to specific
embodiments, it is apparent that other embodiments and variations of this
invention may be devised by others skilled in the art without departing
from the true spirit and scope of the invention. The appended claims are
intended to be construed to include all such embodiments and equivalent
variations.